Articles of manufacture and methods for treatment using adoptive cell therapy

ABSTRACT

Provided are adoptive cell therapy methods involving the administration of doses of cells for treating disease and conditions, including certain B cell malignancies. The cells generally express recombinant receptors such as chimeric antigen receptors (CARs). In some embodiments, the methods are for treating subjects with non-Hodgkin lymphoma (NHL). In some embodiments, the methods are for treating subjects with relapsed or refractory NHL. Also provided are articles of manufacture and prophylactic treatments in connection with adoptive therapy methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

The application is a continuation of U.S. application Ser. No.16/616,938, filed Nov. 25, 2019, which is a National Stage applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2018/035755, filed Jun. 1, 2018, which claims priority from U.S.provisional application No. 62/514,774, filed Jun. 2, 2017, entitled“ARTICLES OF MANUFACTURE AND METHODS FOR TREATMENT USING ADOPTIVE CELLTHERAPY,” U.S. provisional application No. 62/515,530, filed Jun. 5,2017, entitled “ARTICLES OF MANUFACTURE AND METHODS FOR TREATMENT USINGADOPTIVE CELL THERAPY,” U.S. provisional application No. 62/521,366,filed Jun. 16, 2017, entitled “ARTICLES OF MANUFACTURE AND METHODS FORTREATMENT USING ADOPTIVE CELL THERAPY,” U.S. provisional application No.62/527,000, filed Jun. 29, 2017, entitled “ARTICLES OF MANUFACTURE ANDMETHODS FOR TREATMENT USING ADOPTIVE CELL THERAPY,” U.S. provisionalapplication No. 62/549,938, filed Aug. 24, 2017, entitled “ARTICLES OFMANUFACTURE AND METHODS FOR TREATMENT USING ADOPTIVE CELL THERAPY,” U.S.provisional application No. 62/580,425, filed Nov. 1, 2017, entitled“ARTICLES OF MANUFACTURE AND METHODS FOR TREATMENT USING ADOPTIVE CELLTHERAPY,” U.S. provisional application No. 62/593,871, filed Dec. 1,2017, entitled “ARTICLES OF MANUFACTURE AND METHODS FOR TREATMENT USINGADOPTIVE CELL THERAPY,” U.S. provisional application No. 62/596,764,filed Dec. 8, 2017, entitled “ARTICLES OF MANUFACTURE AND METHODS FORTREATMENT USING ADOPTIVE CELL THERAPY,” U.S. provisional application No.62/614,957, filed Jan. 8, 2018, entitled “ARTICLES OF MANUFACTURE ANDMETHODS FOR TREATMENT USING ADOPTIVE CELL THERAPY,” the contents ofwhich are incorporated by reference in their entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled735042012101SeqList.txt, created Jun. 21, 2022, which is 35,389 bytes insize. The information in the electronic format of the Sequence Listingis incorporated by reference in its entirety.

FIELD

The present disclosure relates in some aspects to adoptive cell therapyinvolving the administration of doses of cells for treating subjectswith disease and conditions such as certain B cell malignancies, andrelated methods, compositions, uses and articles of manufacture. Thecells generally express recombinant receptors such as chimeric antigenreceptors (CARs). In some embodiments, the disease or condition is anon-Hodgkin lymphoma (NHL), such as relapsed or refractory NHL orspecific NHL subtype; in some embodiments, the subject is of a specificgroup or subset of NHL subjects, such as heavily pretreated orpoor-prognosis subjects.

BACKGROUND

Various immunotherapy and/or cell therapy methods are available fortreating diseases and conditions. For example, adoptive cell therapies(including those involving the administration of cells expressingchimeric receptors specific for a disease or disorder of interest, suchas chimeric antigen receptors (CARs) and/or other recombinant antigenreceptors, as well as other adoptive immune cell and adoptive T celltherapies) can be beneficial in the treatment of cancer or otherdiseases or disorders. Improved approaches are needed. Provided aremethods and uses that meet such needs.

SUMMARY

Provided herein are methods, uses, compositions, formulations andarticles of manufacture for treating subjects having or suspected ofhaving a disease or condition, such as a cancer or tumor, optionally a Bcell malignancy such as NHL or ALL or CLL or a subtype thereof. Themethods and other embodiments generally relate to administering to thesubject T cells, generally engineered T cells, such as those expressingor containing a recombinant receptor such as a chimeric antigen receptor(CAR) or TCR.

In some embodiments, the dose of cells or cells administered inconnection with any embodiments of the provided methods, compositions,articles of manufacture and uses, contains CD4⁺ T cells or a subtype orphenotype thereof (such as engineered or recombinant receptor-expressingCD4⁺ T cells) and/or CD8⁺ T cells or a subtype thereof (such as anengineered or recombinant receptor-expressing CD4⁺ cells). In someembodiments, the CD8⁺ cells or subtype or phenotype are present at aparticular dose or amount or number; in some embodiments the CD4⁺ cellsor subtype or phenotype are present at a particular dose or amount ornumber. In some embodiments, the CD8⁺ cells or subtype or phenotypethereof and the CD4⁺ cells or subtype or phenotype thereof, are presentin the article or composition or combination, or are administered in themethods, at a defined ratio, such as at or about 1:1, or between at orabout 1:3 and at or about 3:1. In some embodiments, the dose oradministration contains or is of a particular amount or number of onepopulation of the cells and the ratio is a defined ratio or is anaturally-occurring ratio, such as in the blood of the subject fromwhich the cells are derived or ratio that occurs without selection orcontrol for a particular ratio.

In some embodiments, the CD4⁺ T cells (or subset thereof) and the CD8⁺ Tcells (or subset thereof), individually, contain a receptor thatspecifically binds to a target antigen expressed by the disease orcondition, or a cell or tissue thereof, and/or that is associated withthe disease or condition.

In some embodiments, the CD4⁺ and CD8⁺ cells are administered and/orformulated together, e.g. in a single formulation and/or from a singlecontainer.

In some embodiments, separate administrations are carried out of theCD4⁺ and the CD8⁺ cells in the dose, and/or separate formulations orcontainers are included, each individually enriched for the CD4⁺ cellsor the CD4⁺ engineered cells (such as a formulation containing at leasta certain percentage of, e.g., at least 80%, 85%, 90% or 95% or more of,CD4⁺ cells and/or not comprising more than 10% or more than 5% CD8⁺ Tcells) and the CD8⁺ Cells or the CD8⁺ engineered cells (such as aformulation containing at least a certain percentage of, e.g., at least80%, 85%, 90% or 95% or more of, CD8⁺ cells and/or not comprising morethan 10% or more than 5% CD4⁺ T cells).

In some aspects, the administration comprises administering a pluralityof separate compositions, said plurality of separate compositionscomprising a first composition comprising one of the CD4⁺ T cells andthe CD8⁺ T cells and a second composition comprising the other of theCD4⁺ T cells and the CD8⁺ T cells. In certain embodiments of any of theprovided methods, the receptor contained by the CD4⁺ T cells and/or thereceptor contained by the CD8⁺ T cells comprises T cells a recombinantreceptor, and/or wherein the CD4⁺ T cells and/or the CD8⁺ T cells aregenetically engineered to express the receptor.

In some embodiments of any of the provided embodiments, theadministration of the first composition and the administration of thesecond composition are carried out on the same day, are carried outbetween about 0 and about 12 hours apart, between about 0 and about 6hours apart or between about 0 and 2 hours apart; and/or the initiationof administration of the first composition and the initiation ofadministration of the second composition are carried out between about 1minute and about 1 hour apart or between about 5 minutes and about 30minutes apart. In certain embodiments of any of the provided methods,the first composition and second composition are administered no morethan 2 hours, no more than 1 hour, no more than 30 minutes, no more than15 minutes, no more than 10 minutes or no more than 5 minutes apart.

In certain embodiments of any of the provided embodiments, the firstcomposition comprises the CD4⁺ T cells. In some embodiments of any ofthe provided methods, the first composition comprises the CD8⁺ T cells.In particular embodiments of any of the provided methods, the initiationof the administration of the first composition is carried out prior tothe initiation of the administration of the second composition. Incertain embodiments of any of the provided methods, the dose of cellscomprises a defined ratio of CD4⁺ cells expressing a recombinantreceptor to CD8⁺ cells expressing a recombinant receptor and/or of CD4⁺cells to CD8⁺ cells, which ratio optionally is or is approximately 1:1or is between approximately 1:3 and approximately 3:1; and/or the CD4⁺ Tcells comprising the receptor in the one of the first and secondcompositions and the CD8⁺ T cells comprising the receptor in the otherof the first and second compositions are present at a defined ratio,which ratio optionally is or is approximately 1:1 or is betweenapproximately 1:3 and approximately 3:1; and/or the CD4⁺ T cellscomprising the receptor and the CD8⁺ T cells comprising the receptoradministered in the first and second compositions are present at adefined ratio, which ratio optionally is or is approximately 1:1 or isbetween approximately 1:3 and approximately 3:1. In some embodiments ofany of the provided methods, the defined ratio is or is approximately1:1. In particular embodiments of any of the provided methods, the doseof T cells is administered to the subject as a single dose or isadministered only one time within a period of two weeks, one month,three months, six months, 1 year or more. In certain embodiments of anyof the provided methods, the dose of T cells is administered as a doubledose comprising a first dose of the T cells and a consecutive dose ofthe T cells, wherein one or both of the first dose and the second dosecomprises administration of the plurality of compositions of T cells. Insome embodiments of any of the provided methods, the consecutive dose isadministered at a point in time that is at least or more than about 7days or 14 days after and less than about 28 days after initiation ofthe administration of the first dose of cells.

In particular embodiments of any of the provided methods or embodiments,the dose of cells comprises between at or about 1×10⁵ and at or about5×10⁸ total recombinant receptor-expressing T cells or total T cells,between at or about 1×10⁵ and at or about 1×10⁸ total recombinantreceptor-expressing T cells or total T cells, between at or about 5×10⁵and at or about 1×10⁷ total recombinant receptor-expressing T cells ortotal T cells, or from or from about 1×10⁶ to 1×10⁷ total recombinantreceptor-expressing T cells or total T cells, each inclusive. In certainembodiments of any of the provided methods, the dose of T cellscomprises the administration of no more than 1×10⁸ total recombinantreceptor-expressing T cells or total T cells, no more than 1×10⁷ totalrecombinant receptor-expressing T cells or total T cells, no more than0.5×10⁷ total recombinant receptor-expressing T cells or total T cells,no more than 1×10⁶ total recombinant receptor-expressing T cells ortotal T cells, no more than 0.5×10⁶ total recombinantreceptor-expressing T cells or total T cells. In some embodiments of anyof the provided methods, the dose of T cells comprises between at orabout 5×10⁷ recombinant receptor-expressing T cells and 1×10⁸recombinant receptor-expressing T cells, each inclusive.

In particular embodiments of any of the provided methods, therecombinant receptor specifically binds to an antigen associated withthe disease or condition or expressed in cells of the environment of alesion associated with the disease or condition. In certain embodimentsof any of the provided methods, the disease or condition is a cancer. Insome embodiments of any of the provided methods, the disease orcondition is a myeloma, leukemia or lymphoma. In particular embodimentsof any of the provided methods, the antigen is ROR1, B cell maturationantigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (receptortyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, andhepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30,CD33, CD38, CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelialglycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers,EGFR vIII, folate binding protein (FBP), FCRLS, FCRHS, fetalacetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2,kinase insert domain receptor (kdr), kappa light chain, Lewis Y, L1-celladhesion molecule, (L1-CAM), Melanoma-associated antigen (MAGE)-A1,MAGE-A3, MAGE-A6, Preferentially expressed antigen of melanoma (PRAME),survivin, TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3,HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folatereceptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors,5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, a cancer-testesantigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D,NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2,carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor,progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2(OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, GProtein Coupled Receptor 5D (GPCRSD), or a pathogen-specific antigen. Incertain embodiments of any of the provided methods, the antigen is CD19.

In some embodiments of any of the provided methods, the disease orcondition is a B cell malignancy and/or is acute lymphoblastic leukemia(ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkinlymphoma (NHL), and Diffuse Large B-Cell Lymphoma (DLBCL). In particularembodiments of any of the provided methods, the disease or condition isNHL and the NHL is selected from the group consisting of aggressive NHL,diffuse large B cell lymphoma (DLBCL), NOS (not otherwise specified) (denovo and transformed from indolent), primary mediastinal large B celllymphoma (PMBCL), T cell/histocyte-rich large B cell lymphoma (TCHRBCL),Burkitt's lymphoma, mantle cell lymphoma (MCL), and/or follicularlymphoma (FL), optionally follicular lymphoma Grade 3B (FL3B).

In some embodiments of any of the provided methods, the recombinantreceptor includes an extracellular domain containing an antigen-bindingdomain. In some embodiments, the antigen-binding domain is or includesan antibody or an antibody fragment thereof, which optionally is asingle chain fragment. In particular embodiments of any of the providedmethods, the fragment includes antibody variable regions joined by aflexible linker. In some embodiments, the fragment includes an scFv. Insome embodiments of any of the provided methods, the recombinantreceptor also includes a spacer and/or a hinge region.

In certain embodiments of any of the provided methods, the recombinantreceptor includes an intracellular signaling region. In some embodimentsof any of the provided methods, the intracellular signaling regionincludes an intracellular signaling domain. In some embodiments of anyof the provided methods, the intracellular signaling domain is orincludes a primary signaling domain, a signaling domain that is capableof inducing a primary activation signal in a T cell, a signaling domainof a T cell receptor (TCR) component, and/or a signaling domaincontaining an immunoreceptor tyrosine-based activation motif (ITAM). Insome embodiments, the intracellular signaling domain is or includes anintracellular signaling domain of a CD3 chain, optionally a CD3-zeta(CD3ζ) chain, or a signaling portion thereof.

In particular embodiments of any of the provided methods, therecombinant receptor also includes a transmembrane domain disposedbetween the extracellular domain and the intracellular signaling region.

In some embodiments of any of the provided methods, the intracellularsignaling region also includes a costimulatory signaling region. In someembodiments, the costimulatory signaling region includes anintracellular signaling domain of a T cell costimulatory molecule or asignaling portion thereof. In certain embodiments of any of the providedmethods, the costimulatory signaling region includes an intracellularsignaling domain of a CD28, a 4-1BB or an ICOS or a signaling portionthereof. In some embodiments, the costimulatory signaling region isbetween the transmembrane domain and the intracellular signaling region.

In certain embodiments of any of the provided methods, the recombinantreceptor is a chimeric antigen receptor (CAR), optionally wherein therecombinant receptor is a chimeric antigen receptor (CAR), optionallywherein the CAR comprises an extracellular antigen-recognition domainthat specifically binds to the antigen and an intracellular signalingdomain comprising an ITAM, wherein optionally, the intracellularsignaling domain comprises an intracellular domain of a CD3-zeta (CD3ζ)chain; and/or wherein the CAR further comprises a costimulatorysignaling region, which optionally comprises a signaling domain of CD28or 4-1BB.

In some embodiments, the articles of manufacture include a containersuch as a vial comprising a composition comprising CD4⁺ T cellsexpressing a recombinant receptor, and instructions for administering,to a subject having a disease or condition, the composition of CD4⁺ Tcells as a plurality of compositions with a composition comprising CD8⁺T cells expressing a recombinant receptor or a unit dose of cellscomprising all or a portion of the plurality of CD4⁺ T cells and acomposition comprising CD8⁺ T cells expressing a recombinant receptor.In some embodiments, the article of manufacture includes a containersuch as a vial comprising a composition comprising CD8⁺ T cellsexpressing a recombinant receptor, and instructions for administering,to a subject having a disease or condition, the composition of CD8⁺ Tcells as a plurality of compositions with a composition comprising CD4⁺T cells expressing a recombinant receptor or a unit dose of cellscomprising all or a portion of the plurality of CD4⁺ T cells and acomposition comprising CD8⁺ T cells expressing a recombinant receptor.

In some of any of the embodiments, the CAR comprises, in order, the CARincludes an scFv specific for the antigen, a transmembrane domain, acytoplasmic signaling domain derived from a costimulatory molecule,which optionally is or comprises a 4-1BB, and a cytoplasmic signalingdomain derived from a primary signaling ITAM-containing molecule, whichoptionally is or comprises a CD3zeta signaling domain and optionallyfurther includes a spacer between the transmembrane domain and the scFv;

In some of any of the embodiments, the CAR includes, in order, an scFvspecific for the antigen, a transmembrane domain, a cytoplasmicsignaling domain derived from a costimulatory molecule, which optionallyis or comprises a 4-1BB signaling domain, and a cytoplasmic signalingdomain derived from a primary signaling ITAM-containing molecule, whichoptionally is a CD3zeta signaling domain.

In some of any of the embodiments, the CAR comprises or consists of, inorder, an scFv specific for the antigen, a spacer, a transmembranedomain, a cytoplasmic signaling domain derived from a costimulatorymolecule, which optionally is a 4-1BB signaling domain, and acytoplasmic signaling domain derived from a primary signalingITAM-containing molecule, which optionally is or comprises a CD3zetasignaling domain.

In some aspects, the spacer is a polypeptide spacer that (a) comprisesor consists of all or a portion of an immunoglobulin hinge or a modifiedversion thereof or comprises about 15 amino acids or less, and does notcomprise a CD28 extracellular region or a CD8 extracellular region, (b)comprises or consists of all or a portion of an immunoglobulin hinge,optionally an IgG4 hinge, or a modified version thereof and/or comprisesabout 15 amino acids or less, and does not comprise a CD28 extracellularregion or a CD8 extracellular region, or (c) is at or about 12 aminoacids in length and/or comprises or consists of all or a portion of animmunoglobulin hinge, optionally an IgG4, or a modified version thereof;or (d) has or consists of the sequence of SEQ ID NO: 1, a sequenceencoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,SEQ ID NO: 33, SEQ ID NO: 34, or a variant of any of the foregoinghaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto, or (e) comprisesor consists of the formula X₁PPX₂P, where X₁ is glycine, cysteine orarginine and X₂ is cysteine or threonine; and/or the costimulatorydomain comprises SEQ ID NO: 12 or a variant thereof having at least 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity thereto; and/or the primary signaling domaincomprises SEQ ID NO: 13 or 14 or 15 or a variant thereof having at least85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more sequence identity thereto; and/or the scFv comprises a CDRL1sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV(SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37)and/or a CDRH1 sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence ofVIWGSETTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG(SEQ ID NO: 40) or wherein the scFv comprises a variable heavy chainregion of FMC63 and a variable light chain region of FMC63 and/or aCDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence ofFMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3sequence of FMC63 or binds to the same epitope as or competes forbinding with any of the foregoing, and optionally wherein the scFvcomprises, in order, a V_(H), a linker, optionally comprising SEQ ID NO:24, and a V_(L), and/or the scFv comprises a flexible linker and/orcomprises the amino acid sequence set forth as SEQ ID NO: 24.

In some embodiments, the spacer comprises or consists of SEQ ID NO: 1,the costimulatory domain comprises SEQ ID NO: 12 or variant thereofhaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto, the transmembranedomain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof havingat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity thereto, the scFv contains thebinding domain of or CDRs of or V_(H) and V_(L) of FMC63, the primarysignaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variantthereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

In some embodiments, the spacer comprises or consists of SEQ ID NO: 30,the costimulatory domain comprises SEQ ID NO: 12 or variant thereofhaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto, the transmembranedomain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof havingat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity thereto, the scFv contains thebinding domain of or CDRs of or V_(H) and V_(L) of FMC63, the primarysignaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variantthereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

In some embodiments, the spacer comprises or consists of SEQ ID NO: 31,the costimulatory domain comprises SEQ ID NO: 12 or variant thereofhaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto, the transmembranedomain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof havingat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity thereto, the scFv contains thebinding domain of or CDRs of or V_(H) and V_(L) of FMC63, the primarysignaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variantthereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

In some embodiments, the spacer comprises or consists of SEQ ID NO: 33,the costimulatory domain comprises SEQ ID NO: 12 or variant thereofhaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto, the transmembranedomain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof havingat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity thereto, the scFv contains thebinding domain of or CDRs of or V_(H) and V_(L) of FMC63, the primarysignaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variantthereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

In some embodiments, the spacer comprises or consists of SEQ ID NO: 34,the costimulatory domain comprises SEQ ID NO: 12 or variant thereofhaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto, the transmembranedomain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof havingat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity thereto, the scFv contains thebinding domain of or CDRs of or V_(H) and V_(L) of FMC63, the primarysignaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variantthereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.

In some embodiments of any of the provided articles of manufacture, therecombinant receptor expressed by the CD4⁺ cells and the recombinantreceptor expressed by the CD8⁺ T cells is the same or different. Inparticular embodiments of any of the provided articles of manufacture,vial comprises greater than or greater than about 10×10⁶ T cells orrecombinant receptor-expressing T cells, greater than or greater thanabout 15×10⁶ T cells or recombinant receptor-expressing T cells, greaterthan or greater than about 25×10⁶ T cells or recombinantreceptor-expressing T cell.

In certain embodiments of any of the provided articles of manufacture,the vial comprises between about 10 million cells per ml and about 70million cells per ml, between about 10 million cells per ml and about 50million cells per ml, between about 10 million cells per ml and about 25million cells per ml, between about 10 million cells per ml and about 15million cells per ml, 15 million cells per ml and about 70 million cellsper ml, between about 15 million cells per ml and about 50 million cellsper ml, between about 15 million cells per ml and about 25 million cellsper ml, between about 25 million cells per ml and about 70 million cellsper ml, between about 25 million cells per ml and about 50 million cellsper ml, and between about 50 million cells per ml and about 70 millioncells per ml. In some embodiments of any of the provided articles ofmanufacture, the composition further comprises a cryoprotectant and/orthe article further includes instructions for thawing the compositionprior to administration to the subject.

In some embodiments of any of the provided articles of manufacture, thecompositions or plurality of compositions comprises a dose of cellscomprising from or from about 2×10⁷ to about 4×10⁷ CD8⁺ cells, such asabout 2×10⁷, 2.5×10⁷, 3×10⁷, 3.5×10⁷, or 4×10⁷ CD8⁺ cells, and from orfrom about 2×10⁷ to about 4×10⁷ CD4⁺ cells, such as about 2×10⁷,2.5×10⁷, 3×10⁷, 3.5×10⁷, or 4×10⁷ CD4⁺ cells, each inclusive. In someembodiments, the compositions or plurality of compositions comprises adose of cells comprising approximately 3×10⁷ CD8⁺ cells and 3.5×10⁷ CD4⁺cells.

In particular embodiments of any of the provided articles ofmanufacture, the plurality of compositions of cells comprises a definedratio of CD4⁺ cells expressing the recombinant receptor to CD8⁺ cellsexpressing the recombinant receptor and/or of CD4⁺ cells to CD8⁺ cells,which ratio optionally is approximately 1:1 or is between approximately1:3 and approximately 3:1. In certain embodiments of any of the providedarticles of manufacture, the defined ratio is or is approximately 1:1.In some embodiments of any of the provided articles of manufacture, theplurality of compositions, collectively, comprises a dose of cellscomprising from or from about 1×10⁵ to 5×10⁸ total recombinantreceptor-expressing T cells or total T cells, 1×10⁵ to 1×10⁸ totalrecombinant receptor-expressing T cells or total T cells, from or fromabout 5×10⁵ to 1×10⁷ total recombinant receptor-expressing T cells ortotal T cells, or from or from about 1×10⁶ to 1×10⁷ total recombinantreceptor-expressing T cells or total T cells, each inclusive. Inparticular embodiments of any of the provided articles of manufacture,the plurality of compositions, collectively, comprises a dose of cellscomprising no more than 1×10⁸ total recombinant receptor-expressing Tcells or total T cells, no more than 1×10⁷ total recombinantreceptor-expressing T cells or total T cells, no more than 0.5×10⁷ totalrecombinant receptor-expressing T cells or total T cells, no more than1×10⁶ total recombinant receptor-expressing T cells or total T cells, nomore than 0.5×10⁶ total recombinant receptor-expressing T cells or totalT cells. In certain embodiments of any of the provided articles ofmanufacture, the plurality of compositions, collectively, comprises adose of cells comprising between at or about 5×10⁷ recombinantreceptor-expressing T cells and 1×10⁸ recombinant receptor-expressing Tcells, each inclusive.

In some embodiments of any of the provided articles of manufacture, theinstructions specify administering the composition comprising the CD4⁺ Tcells and the composition comprising the CD8⁺ T cells 0 to 12 hoursapart, 0 to 6 hours apart or 0 to 2 hours apart. In particularembodiments of any of the provided articles of manufacture, theinstructions specify administering the composition comprising the CD4⁺ Tcells and the composition comprising the CD8⁺ T cells no more than 2hours, no more than 1 hour, no more than 30 minutes, no more than 15minutes, no more than 10 minutes or no more than 5 minutes apart. Incertain embodiments of any of the provided articles of manufacture, theinstructions specify administering the composition comprising the CD4⁺ Tcells prior to administering the composition comprising the CD8⁺ cells.In some embodiments of any of the provided articles of manufacture, theinstructions specify administering the composition comprising the CD8⁺ Tcells prior to administering the composition comprising the CD4⁺ cells.

In particular embodiments of any of the provided articles ofmanufacture, the recombinant receptor specifically binds to an antigenassociated with the disease or condition or expressed in cells of theenvironment of a lesion associated with the disease or condition. Incertain embodiments of any of the provided articles of manufacture, thedisease or condition is a cancer. In some embodiments of any of theprovided articles of manufacture, the disease or condition is a myeloma,leukemia or lymphoma.

In particular embodiments of any of the provided articles ofmanufacture, the antigen is ROR1, B cell maturation antigen (BCMA),carbonic anhydrase 9 (CAIX), tEGFR, Her2/neu (receptor tyrosine kinaseerbB2), L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis Bsurface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38,CD44, EGFR, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII,folate binding protein (FBP), FCRLS, FCRHS, fetal acetylcholinereceptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insertdomain receptor (kdr), kappa light chain, Lewis Y, L1-cell adhesionmolecule, (L1-CAM), Melanoma-associated antigen (MAGE)-A1, MAGE-A3,MAGE-A6, Preferentially expressed antigen of melanoma (PRAME), survivin,TAG72, B7-H6, IL-13 receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA,CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folatereceptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors,5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, a cancer-testesantigen, mesothelin, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D,NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2,carcinoembryonic antigen (CEA), Her2/neu, estrogen receptor,progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2(OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, GProtein Coupled Receptor 5D (GPCRSD), or a pathogen-specific antigen. Incertain embodiments of any of the provided articles of manufacture, theantigen is CD19.

In some embodiments of any of the provided articles of manufacture, thedisease or condition is a B cell malignancy and/or is acutelymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia(CLL), non-Hodgkin lymphoma (NHL), and Diffuse Large B-Cell Lymphoma(DLBCL). In particular embodiments of any of the provided articles ofmanufacture, the disease or condition is NHL and the NHL is selectedfrom the group consisting of aggressive NHL, diffuse large B celllymphoma (DLBCL), NOS (de novo and transformed from indolent), primarymediastinal large B cell lymphoma (PMBCL), T cell/histocyte-rich large Bcell lymphoma (TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL),and/or follicular lymphoma (FL), optionally follicular lymphoma Grade 3B(FL3B).

In certain embodiments of any of the provided articles of manufacture,the T cells are primary T cells obtained from a subject. In someembodiments of any of the provided articles of manufacture, the T cellsare autologous to the subject. In particular embodiments of any of theprovided articles of manufacture, the T cells are allogeneic to thesubject.

In certain embodiments of any of the provided articles of manufacture,the recombinant receptor is or includes a functional non-TCR antigenreceptor or a TCR or antigen-binding fragment thereof. In someembodiments, the recombinant receptor is a chimeric antigen receptor(CAR).

In particular embodiments any of the provided articles of manufacture,the recombinant receptor includes an extracellular domain containing anantigen-binding domain. In some embodiments, the antigen-binding domainis or includes an antibody or an antibody fragment thereof, whichoptionally is a single chain fragment. In some embodiments, the fragmentincludes antibody variable regions joined by a flexible linker. In someembodiments, the fragment includes an scFv.

In some embodiments any of the provided articles of manufacture ormethods, the recombinant receptor also includes a spacer and/or a hingeregion.

In certain embodiments any of the provided articles of manufacture ormethods, the recombinant receptor includes an intracellular signalingregion. In some embodiments, the intracellular signaling region includesan intracellular signaling domain. In some embodiments any of theprovided articles of manufacture, the intracellular signaling domain isor includes a primary signaling domain, a signaling domain that iscapable of inducing a primary activation signal in a T cell, a signalingdomain of a T cell receptor (TCR) component, and/or a signaling domaincontaining an immunoreceptor tyrosine-based activation motif (ITAM). Insome embodiments, the intracellular signaling domain is or includes anintracellular signaling domain of a CD3 chain, optionally a CD3-zeta(CD3ζ) chain, or a signaling portion thereof.

In particular embodiments any of the provided articles of manufacture ormethods, the recombinant receptor also includes a transmembrane domaindisposed between the extracellular domain and the intracellularsignaling region.

In some embodiments any of the provided articles of manufacture ormethods, the intracellular signaling region also includes acostimulatory signaling region. In some embodiments, the costimulatorysignaling region includes an intracellular signaling domain of a T cellcostimulatory molecule or a signaling portion thereof. In someembodiments any of the provided articles of manufacture, thecostimulatory signaling region includes an intracellular signalingdomain of a CD28, a 4-1BB or an ICOS or a signaling portion thereof. Insome embodiments, the costimulatory signaling region is between thetransmembrane domain and the intracellular signaling region.

In some embodiments, the methods and articles are for or capable oftreating a subject having non-Hodgkin lymphoma (NHL). In some aspects,the method involves, and/or the article of manufacture specifies orincludes formulations capable of, administering to the subject a dose orplurality of T cells. In some aspects, the T cells comprising T cellssuch as CD8⁺ T cells and/or CD4⁺ T cells, expressing a chimeric antigenreceptor (CAR) that specifically binds to a target antigen expressed bythe NHL.

In some embodiments, the dose of T cells comprises between at or about5×10⁷ CAR-expressing T cells and 1×10⁸ CAR-expressing T cells,inclusive; and the NHL comprises diffuse large B cell lymphoma (DLBCL),primary mediastinal large B cell lymphoma (PMBCL), NOS (de novo ortransformed from indolent lymphoma), or follicular lymphoma Grade 3B andwherein the subject is or has been identified as having an EasternCooperative Oncology Group Performance Status (ECOG) status of 0 or 1.

In some embodiments of any of the provided embodiments, the methodsfurther comprise identifying, or the article of manufacture includesinformation specifying, treatment of a subject having diffuse large Bcell lymphoma (DLBCL), primary mediastinal large B cell lymphoma(PMBCL), NOS (de novo or transformed from indolent lymphoma), orfollicular lymphoma Grade 3B having an ECOG status of 0 or 1. In someembodiments of any of the provided embodiments, the dose of T cellsand/or the cells administered comprises a defined ratio of CD4⁺ cellsexpressing the CAR to CD8⁺ cells expressing the CAR and/or of CD4⁺ cellsto CD8⁺ cells, which ratio optionally is approximately 1:1 or is betweenapproximately 1:3 and approximately 3:1.

In some aspects, the embodiments are for treating a subject havingnon-Hodgkin lymphoma (NHL) and involve administering to the subject adose of T cells comprising T cells expressing a chimeric antigenreceptor (CAR) that specifically binds to a target antigen expressed bythe NHL. In some aspects, the dose of T cells includes a defined ratioof CD4⁺ cells expressing the CAR to CD8⁺ cells expressing the CAR and/orof CD4⁺ cells to CD8⁺ cells, which ratio in some aspects isapproximately or is 1:1. In some aspects, the NHL comprises diffuselarge B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma(PMBCL), NOS (not otherwise specified) (de novo or transformed fromindolent lymphoma), or follicular lymphoma Grade 3B.

In particular embodiments of any of the provided embodiments, thesubject is or has been identified as or is specified as having anEastern Cooperative Oncology Group Performance Status (ECOG) status of0, 1 or 2. In certain embodiments of any of the provided embodiments,the subject is or has been identified as or is specified as having anECOG status of 0 or 1.

In some embodiments of any of the provided embodiments, the methodsand/or uses and/or administration of cells according to the articles ofmanufacture, achieve certain outcomes and/or are associated with certainreduced risks of toxicity, e.g., in the population of subjects treatedaccording to the methods or according to information provided in thearticle of manufacture. In some aspects: at least 35%, at least 40% orat least 50% of subjects treated according to the method achieve acomplete response (CR) and/or a durable CR; and/or at least 50%, atleast 60% or at least 70% of the subjects treated according to themethod achieve objective response (OR) and/or a durable OR. Inparticular embodiments of any of the provided methods, the response isdurable for greater than 3 months or greater than 6 months. In someembodiments, at least 40%, at least 50%, at least 60%, at least 70% ofthe subjects who, at or prior to the administration of the dose of cellshad or were identified to have a double/triple hit lymphoma or relapse,optionally relapse within 12 months, following administration of anautologous stem cell transplant (ASCT), achieved an OR, optionallywherein the OR is durable for at or greater than 3 months or at orgreater than 6 months. In certain embodiments of any of the providedmethods, greater than or greater than about 50% of the subjects treatedaccording to the method do not exhibit a grade 3 or greater cytokinerelease syndrome (CRS) or a grade 3 or greater neurotoxicity. In someembodiments, such subjects do not exhibit early onset CRS and/orneurotoxicity.

Provided herein are methods of assessing likelihood of a response to acell therapy, the methods involving: assessing the level, amount orconcentration of one or more analyte in a biological sample, wherein theone or more analyte is selected from ferritin, LDH, CXCL10, G-CSF, andIL-10, wherein: the biological sample is from a subject that is acandidate for treatment with the cell therapy, said cell therapycomprising a dose of genetically engineered cells expressing arecombinant receptor; and the biological sample is obtained from thesubject prior to administering the cell therapy and/or said biologicalsample does not comprise the recombinant receptor and/or said engineeredcells; and comparing, individually, the level, amount or concentrationof the analyte in the sample to a threshold level, thereby determining alikelihood that a subject will achieve a response to the cell therapy.In some embodiments, the methods also involve administering the celltherapy to the subject if the subject is likely to achieve a response.

Provided herein are methods of selecting a subject for treatment, themethods involving: assessing the level, amount or concentration of oneor more analyte in a biological sample, wherein the one or more analyteis selected from ferritin, LDH, CXCL10, G-CSF, and IL-10, wherein: thebiological sample is from a subject that is a candidate for treatmentwith the cell therapy, said cell therapy comprising a dose ofgenetically engineered cells expressing a recombinant receptor; and thebiological sample is obtained from the subject prior to administeringthe cell therapy and/or said biological sample does not comprise therecombinant receptor and/or said engineered cells; and selecting asubject who is likely to respond to treatment based on the results ofdetermining a likelihood that a subject will achieve a response to thecell therapy by comparing, individually, the level, amount orconcentration of the analyte in the sample to a threshold level. In someembodiments, the methods also involve administering the cell therapy tothe subject selected for treatment.

Provided herein are methods of treatment, the methods involving:selecting a subject who is likely to respond to treatment with a celltherapy based on the results of determining a likelihood that a subjectwill achieve a response to the cell therapy by comparing, individually,the level, amount or concentration of one or more analyte in abiological sample, wherein the one or more analyte is selected fromferritin, LDH, CXCL10, G-CSF, and IL-10, to a threshold level, wherein:the biological sample is from a subject that is a candidate fortreatment with the cell therapy, said cell therapy comprising a dose ofgenetically engineered cells expressing a recombinant receptor; and thebiological sample is obtained from the subject prior to administeringthe cell therapy and/or said biological sample does not comprise therecombinant receptor and/or said engineered cells; and administering thecell therapy to a subject selected for treatment.

In some embodiments, the subject is likely to achieve a response if thelevel, amount or concentration of one or more of the analyte is below athreshold level and the subject is not likely to achieve a response ifthe level, amount or concentration of one or more of the analyte isabove a threshold level.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 10% or within 5% and/or is within a standarddeviation below the median or mean level, amount or concentration, or isor is about the median or mean level, amount or concentration, of theanalyte in a biological sample obtained from a group of subjects priorto receiving a cell therapy, wherein each of the subjects of the groupwent on to achieve a response after administration of arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 10% or within 5% and/or is within a standarddeviation above the median or mean level, amount or concentration of theanalyte in a biological sample obtained from a group of subjects priorto receiving a cell therapy, wherein each of the subjects of the groupwent on to exhibit stable disease (SD) and/or progressive disease (PD)after administration of a recombinant-receptor-expressing therapeuticcell composition for treating the same disease or condition.

In some embodiments, the response comprises objective response. In someembodiments, the objective response comprises complete response (CR) orpartial response (PR).

Provided herein are methods of assessing likelihood of a durableresponse to a cell therapy, the methods involving: assessing the level,amount or concentration of one or more analyte in a biological sample,wherein the one or more analyte is selected from LDH, ferritin, CRP,D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α,CXCL-10, IL-8, MCP-1 and MIP-1β, wherein: the biological sample is froma subject that is a candidate for treatment with the cell therapy, saidcell therapy comprising a dose of genetically engineered cellsexpressing a recombinant receptor; and the biological sample is obtainedfrom the subject prior to administering the cell therapy and/or saidbiological sample does not comprise the recombinant receptor and/or saidengineered cells; and comparing, individually, the level, amount orconcentration of the analyte in the sample to a threshold level, therebydetermining a likelihood that a subject will achieve a durable responseto the cell therapy.

In some embodiments, the methods also involve administering the celltherapy to the subject if the subject is likely to achieve a response.

Provided herein are methods of selecting a subject for treatment, themethods involving: assessing the level, amount or concentration of oneor more analyte in a biological sample, wherein the one or more analyteis selected from LDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15,IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β, wherein:the biological sample is from a subject that is a candidate fortreatment with the cell therapy, said cell therapy comprising a dose ofgenetically engineered cells expressing a recombinant receptor; and thebiological sample is obtained from the subject prior to administeringthe cell therapy and/or said biological sample does not comprise therecombinant receptor and/or said engineered cells; and selecting asubject who is likely to respond to treatment based on the results ofdetermining a likelihood that a subject will achieve a durable responseto the cell therapy by comparing, individually, the level, amount orconcentration of the analyte in the sample to a threshold level. In someembodiments, the methods also involve administering the cell therapy tothe subject selected for treatment.

Provided herein are methods of treatment, the methods involving:selecting a subject who is likely to respond to treatment with a celltherapy based on the results of determining a likelihood that a subjectwill achieve a durable response to the cell therapy by comparing,individually, the level, amount or concentration of one or more analytein a biological sample to a threshold level, wherein the one or moreanalyte is selected from LDH, ferritin, CRP, D-dimer, SAA-1, IL-6,IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 andMIP-1β, wherein: the biological sample is from a subject that is acandidate for treatment with the cell therapy, said cell therapycomprising a dose of genetically engineered cells expressing arecombinant receptor; and the biological sample is obtained from thesubject prior to administering the cell therapy and/or said biologicalsample does not comprise the recombinant receptor and/or said engineeredcells; and administering the cell therapy to a subject selected fortreatment.

In some embodiments, the subject is likely to achieve a durable responseif the level, amount or concentration one or more of the analyte isbelow a threshold level and the subject is not likely to achieve adurable response if the level, amount or concentration one or more ofthe analyte is above a threshold level.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 10% or within 5% and/or is within a standarddeviation below the median or mean level, amount or concentration, or isor is about the median or mean level, amount or concentration, of theanalyte in a biological sample obtained from a group of subjects priorto receiving a cell therapy, wherein each of the subjects of the groupwent on to achieve a durable response after administration of arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 11% or within 5% and/or is within a standarddeviation above the median or mean level, amount or concentration of theanalyte in a biological sample obtained from a group of subjects priorto receiving a cell therapy, wherein each of the subjects of the groupdid not achieve a durable response after administration of arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments, the durable response comprises a complete response(CR) or partial response (PR) that is durable for at or greater than 3months, 4 months, 5 months, or 6 months.

In some embodiments, the durable response comprises a CR or PR that isdurable for at least 3 months.

Provided herein are methods of assessing the risk of developing atoxicity after administration of a cell therapy, the methods involvingassessing the level, amount or concentration of one or more analyte in abiological sample from a subject or a volumetric measure of tumor burdenin a subject, wherein the one or more analyte is selected from LDH,Ferritin, C-reactive protein (CRP), D-dimer (fibrin degradationproduct), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP-1αand MIP-1β, wherein: the subject is a candidate for treatment with thecell therapy, said cell therapy optionally comprising a dose orcomposition of genetically engineered cells expressing a recombinantreceptor; and the biological sample is obtained from the subject priorto administering the cell therapy and/or said biological sample does notcomprise the recombinant receptor and/or said engineered cells; andcomparing, individually, the level, amount or concentration of theanalyte in the sample or the volumetric measure of tumor burden to athreshold level, thereby determining a risk of developing a toxicityafter administration of the cell therapy.

Provided herein are methods of identifying a subject, the methodsinvolving assessing the level, amount or concentration of one or moreanalyte in a biological sample from a subject or a volumetric measure oftumor burden in a subject, wherein the one or more analyte is selectedfrom LDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrindegradation product), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2,MCP-1, MIP-1α and MIP-1β, wherein: the subject is a candidate fortreatment with the cell therapy, said cell therapy optionally comprisinga dose or composition of genetically engineered cells expressing arecombinant receptor; and the biological sample is obtained from thesubject prior to administering the cell therapy and/or said biologicalsample does not comprise the recombinant receptor and/or said engineeredcells; and identifying a subject who has a risk of developing a toxicityafter administration of a cell therapy based by comparing, individually,the level, amount or concentration of the analyte in the sample or thevolumetric measure of tumor burden to a threshold level.

Provided herein are methods of treatment, comprising assessing thelevel, amount or concentration of one or more analyte in a biologicalsample from a subject or a volumetric measure of tumor burden in thesubject, wherein the one or more analyte is selected from LDH, Ferritin,C-reactive protein (CRP), D-dimer (fibrin degradation product), IL-6,IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP-1α and MIP-1β,wherein: the subject is a candidate for treatment with the cell therapy,said cell therapy optionally comprising a dose or composition ofgenetically engineered cells expressing a recombinant receptor; and thebiological sample is obtained from the subject prior to administeringthe cell therapy and/or said biological sample does not comprise therecombinant receptor and/or said engineered cells; and; and comparing,individually, the level, amount or concentration of the analyte in thesample or the volumetric measure of tumor burden to a threshold level,thereby determining a risk of developing a toxicity after administrationof the cell therapy; and following or based on the results of theassessment, administering to the subject the cell therapy, and,optionally, an agent or other treatment capable of treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity.

In some embodiments, the biological sample is a blood or plasma sample.

In some embodiments, the volumetric measure of tumor burden is a sum ofproduct dimensions (SPD) or is a volumetric measurement based on CTand/or MM imaging or other imaging of body. In some embodiments, thevolumetric measure of tumor burden is carried out prior to treatment,prior to apheresis, or prior to cell product manufacturing.

In some embodiments, the methods also involve monitoring the subject forsymptoms of toxicity if the subject is administered a cell therapy andis identified as having a risk of developing a toxicity.

In some embodiments, the subject has a risk of developing a toxicity ifthe level, amount or concentration one or more of the analyte or thevolumetric measure of tumor burden is above a threshold level and thesubject has a low risk of developing a toxicity if the level, amount orconcentration one or more of the analyte or the volumetric measure oftumor burden is below a threshold level.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 10% or within 5% and/or is within a standarddeviation above the median or mean level, amount or concentration, or isor is about the median or mean level, amount or concentration, of theanalyte or the volumetric measure of tumor burden in a biological sampleobtained from a group of subjects prior to receiving a cell therapy,wherein each of the subjects of the group went on not to develop anytoxicity after receiving a recombinant-receptor-expressing therapeuticcell composition for treating the same disease or condition.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 10% or within 5% and/or is within a standarddeviation below the median or mean level, amount or concentration of theanalyte or the volumetric measure of tumor burden in a biological sampleobtained from a group of subjects prior to receiving a cell therapy,wherein each of the subjects of the group went on to develop a toxicityafter receiving a recombinant-receptor-expressing therapeutic cellcomposition for treating the same disease or condition.

In some embodiments, the toxicity is neurotoxicity or CRS.

In some embodiments, the toxicity is grade 1 or higher neurotoxicity orCRS.

In some embodiments, the toxicity is severe neurotoxicity or is grade 2or higher neurotoxicit, a grade 3 or higher neurotoxicity, at leastprolonged grade 3 neurotoxicity or is at or above grade 4 or grade 5neurotoxixity; or the toxicity is severe CRS or comprises grade 2 orhigher or grade 3 or higher CRS.

In some embodiments, the toxicity is neurotoxicity and the volumetricmeasure of tumor burden is SPD and the one or more analyte is selectedfrom LDH, IL-10, IL-15, IL-16, TNF-α and MIP-1β.

In some embodiments, the toxicity is neurotoxicity and one or moreanalytes is assessed and the analytes are selected from LDH, Ferritin,CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 and MIP-1β.

In some embodiments, the toxicity is neurotoxicity and one or moreanalytes is assessed and the analytes are selected from IL-8, IL-10 andCXCL10.

In some embodiments, the neurotoxicity is severe neurotoxicity or grade3 or higher neurotoxicity.

In some embodiments, toxicity is CRS and the one or more analyte orvolumetric measure of tumor burden is selected from LDH, SPD, CRP,d-dimer, IL-6, IL-15, TNF-α and MIP-1α.

In some embodiments, the CRS is severe CRS or grade 3 or higher CRS.

In some embodiments, if the subject is identified as having a risk ofdeveloping a toxicity, administering to the subject: (1) an agent orother treatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity and (2)the cell therapy, wherein administration of the agent is to beadministered (i) prior to, (ii) within one, two, or three days of, (iii)concurrently with and/or (iv) at first fever following, the initiationof administration of the cell therapy to the subject; and/or a celltherapy at a reduced dose or at a dose that is not associated with riskof developing toxicity or severe toxicity, or is not associated with arisk of developing a toxicity or severe toxicity in a majority ofsubjects, and/or a majority of subjects having a disease or conditionthat the subject has or is suspected of having, following administrationof the cell therapy; and/or administering to the subject a cell therapyin an in-patient setting and/or with admission to the hospital for oneor more days, optionally wherein the cell therapy is otherwise to beadministered to subjects on an outpatient basis or without admission tothe hospital for one or more days.

In some embodiments, the agent or other treatment is an anti-IL-6antibody or an anti-IL6 receptor antibody.

In some embodiments, the agent or other treatment is or comprises anagent selected from among tocilizumab, siltuximab, clazakizumab,sarilumab, olokizumab (CDP6038), elsilimomab, ALD518/BMS-945429,sirukumab (CNTO 136), CPSI-2634, ARGX-109, FE301 and FM101.

In some embodiments, the agent or other treatment is or comprises asteroid, optionally dexamethasone.

In some embodiments, a volumetric measure is assessed and the volumetricmeasure is SPD and the threshold level is or is about 30 cm², is or isabout 40 cm², is or is about 50 cm², is or is about 60 cm², or is or isabout 70 cm². In some embodiments, the volumetric measure is SPD and thethreshold level is or is about 50 cm².

In some embodiments, the one or more analyte is or comprises LDH and thethreshold level is or is about 300 units per liter, is or is about 400units per liter, is or is about 500 units per liter or is or is about600 units per liter. In some embodiments, the analyte is LDH and thethreshold level is or is about 500 units per liter.

In some embodiments, the recombinant receptor specifically binds to anantigen associated with the disease or condition or expressed in cellsof the environment of a lesion associated with the disease or condition.In some embodiments, the disease or condition is a cancer. In someembodiments, the disease or condition is a myeloma, leukemia orlymphoma. In some embodiments, the disease or condition is a B cellmalignancy and/or is acute lymphoblastic leukemia (ALL), adult ALL,chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), andDiffuse Large B-Cell Lymphoma (DLBCL).

In some embodiments, the recombinant receptor is a chimeric antigenreceptor (CAR). In some embodiments, the engineered cells comprise Tcells, optionally CD4⁺ and/or CD8⁺. In some embodiments, the T cells areprimary T cells obtained from a subject or are autologous to thesubject.

Provided herein are methods of treating a subject having non-Hodgkinlymphoma (NHL), the methods comprising administering to the subject adose of T cells comprising T cells expressing a chimeric antigenreceptor (CAR) that specifically binds to a target antigen expressed bythe NHL, wherein: the dose of T cells comprises between at or about5×10⁷ recombinant receptor-expressing T cells and 1×10⁸ recombinantreceptor-expressing T cells, inclusive, said dose comprising a definedratio of CD4⁺ cells expressing the recombinant receptor to CD8⁺ cellsexpressing the recombinant receptor and/or of CD4⁺ cells to CD8⁺ cells,which ratio is approximately or is 1:1; and the method results in (1) acomplete response (CR) in at least 35%, at least 40% or at least 50% ofsubjects treated and/or objective response (OR) in at least 50%, atleast 60% or at least 70% of subjects treated and (2) results in no morethan 50% of subjects exhibiting a cytokine release syndrome (CRS) higherthan grade 2 and/or a neurotoxicity higher than grade 2.

In some embodiments of any of the provided methods, at least 40%, atleast 50%, at least 60%, at least 70% of the subjects who, at or priorto the administration of the dose of cells had or were identified tohave a double/triple hit lymphoma (or high-grade B-cell lymphoma, withMYC and BCL2 and/or BCL6 rearrangements with DLBCL histology(double/triple hit)) or relapse following administration of anautologous stem cell transplant (ASCT), achieved an OR, optionallywherein the OR is durable for at or greater than 3 months or at orgreater than 6 months.

In some embodiments of any of the provided methods, the CR or the OR isdurable for greater than 3 months or greater than 6 months. Inparticular embodiments of any of the provided methods, greater than orgreater than about 50% of the subjects treated according to the methoddo not exhibit any grade of cytokine release syndrome (CRS) orneurotoxicity.

In some embodiments, the CR or the OR is durable for greater than 3months or greater than 6 months; at least 20%, at least 25%, at least35%, at least 40% or at least 50% of subjects treated according to themethod achieve a CR that is durable; at least 60%, 70%, 80%, 90%, or 95%of subjects treated with the method and who achieve a CR, remain in CRor remain in response or remain surviving for at or greater than 3months or at or greater than 6 months or at or greater than 9 months;and/or wherein at least 60%, 70%, 80%, 90%, or 95% of subjects treatedwith the method who achieve a CR by one month and/or by three monthsremain in response, remain in CR, and/or survive or survive withoutprogression, for greater at or greater than 3 months and/or at orgreater than 6 months and/or at greater than nine months; and/or atleast 50%, at least 60% or at least 70% of the subjects treatedaccording to the method achieve objective response (OR) optionallywherein the OR is durable, or is durable in at least 60%, 70%, 80%, 90%,or 95% of subjects achieving the OR, for at or greater than 3 months orat or greater than 6 months; and/or wherein at least 60%, 70%, 80%, 90%,or 95% of subjects treated with the method and achieving an OR remain inresponse or surviving for greater at or greater than 3 months and/or ator greater than 6 months.

In some embodiments, at or prior to administration of the dose of cells,the subject is or has been identified as having a lymphoma associatedwith or involving central nervous system (CNS) involvement; and/or atleast 70%, at least 80%, at least 90% or at least 95% of subjectstreated according to the method who, at or prior to the administrationof the dose of cells exhibited or were identified to exhibit a lymphomawith CNS involvement, achieved a resolution of the CNS disease.

Provided herein are methods of treating a subject, the method involvingadministering, to a subject that has a lymphoma a dose of T cellscomprising T cells expressing a chimeric antigen receptor (CAR) thatspecifically binds to a target antigen expressed by the lymphoma,wherein the lymphoma in the subject is associated with or involvescentral nervous system (CNS) involvement. In some aspects, at or priorto the time of administration of the dose of cells, the subjectcomprises a brain lesion, optionally a temporal lobe brain lesion. Insome examples, the lymphoma is a B cell malignancy. In some embodiments,the lymphoma is non-Hodgkin lymphoma (NHL).

In some of any such embodiments, at least 35%, at least 40% or at least50% of subjects treated according to the method achieve a completeresponse (CR) or remission of CNS disease and/or achieve reduction in orclearance of CNS disease, optionally wherein the CR or remission orreduction or clearance of the CNS disease is durable, or is durable inat least 60%, 70%, 80%, 90%, or 95% of subjects achieving the CR, for ator greater than 3 months or at or greater than 6 months; and/or at least60%, 70%, 80%, 90%, or 95% of subjects achieving a CR or remission orother reduction of CNS disease by one month and/or by three monthsremain in response, remain in remission, e.g., in CR, or remain showingsigns of the reduction or remission, and/or survive or survive withoutprogression, for greater at or greater than 3 months and/or at orgreater than 6 months and/or at greater than nine months; and/or atleast 50%, at least 60% or at least 70% of the subjects treatedaccording to the method achieve objective response (OR) or remission ofCNS disease optionally wherein the OR or remission of the CNS disease isdurable, or is durable in at least 60%, 70%, 80%, 90%, or 95% ofsubjects achieving the OR, for at or greater than 3 months or at orgreater than 6 months; and/or at least 60%, 70%, 80%, 90%, or 95% ofsubjects achieving the OR or remission of CNS disease remain in responseor surviving for greater at or greater than 3 months and/or at orgreater than 6 months; and/or the brain lesion is reduced in size orvolume, optionally by greater than or greater than about 25%, 50%, 75%or more. In some aspects, reduction or remission or clearance of CNSdisease is achieved without or without substantial signs or symptoms ofa toxicity, such as a neurotoxicity such as severe neurotoxicity, e.g.,neurotoxicity greater than grade 2 or greater than grade 3, and/orwithout toxicity caused by activation or presence of the cellulartherapy cells in the brain of the subject, and/or is achieved without anincreased level of the toxicity, as compared to a subject in which CNSdisease remains and/or treated with the therapy but that does notexhibit CNS disease.

In some embodiments of any of the provided methods, greater than orgreater than about 30%, 35%, 40%, or 50% of the subjects treatedaccording to the method do not exhibit any grade of cytokine releasesyndrome (CRS) or neurotoxicity. In some embodiments of any of theprovided methods, at least at or about 45%, 50%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of subjects treated according to the method do notexhibit early onset CRS or neurotoxicity and/or do not exhibit onset ofCRS earlier than 3 days following initiation of the administrationand/or do not exhibit onset of neurotoxicity earlier than 5 daysfollowing initiation of the administration and/or wherein the medianonset of neurotoxicity among subjects treated according to the method isat or after the median peak of, or median time to resolution of, CRS insubjects treated according to the method and/or the median onset ofneurotoxicity among subjects treated according to the method is greaterthan at or about 8, 9, 10, or 11 days.

In certain embodiments of any of the provided methods, prior toinitiation of administration of the dose of cells, the subject has notbeen administered an agent or treatment to capable of treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity. In certain embodiments of any of the providedmethods, the subject is not administered an agent or treatment for thetreatment or prevention or reduction or attenuation of a neurotoxicityand/or a cytokine release syndrome or risk thereof, within a period oftime following administration of the dose, which period of time isoptionally at or about 1, 2, 3, 4, 5 days or is optionally at or about6, 7, 8, 9, 10, 11 days or is optionally 1, 2, 3 or 4 weeks. In certainembodiments of any of the provided methods, the subject is notadministered an agent or treatment for the treatment or prevention orreduction or attenuation of a neurotoxicity and/or a cytokine releasesyndrome or risk thereof, following administration of the dose, prior toor unless the subject exhibits a sign or symptom of the toxicity and/orprior to or unless the subject exhibits a sign or symptom of thetoxicity other than a fever, optionally wherein the fever is not asustained fever or the fever is or has been reduced or reduced by morethan 1° C. after treatment with an antipyretic. In certain embodimentsof any of the provided methods, the administration and any follow-up iscarried out on an outpatient basis and/or without admitting the subjectto a hospital and/or without an overnight stay at a hospital and/orwithout requiring admission to or an overnight stay at a hospital,optionally unless or until the subject exhibits a sustained fever or afever that is or has not been reduced or not reduced by more than 1° C.after treatment with an antipyretic.

In some embodiments of any of the provided methods, prior to initiationof administration of the dose of cells, the subject has not beenadministered an anti-IL-6 or anti-IL-6R antibody, optionally tocilizumabor situximab, and/or has not been administered a steroid, optionallydexamethasone. In certain embodiments of any of the provided methods,the subject is not administered an anti-IL-6 or anti-IL-6R antibody,optionally tocilizumab or siltuximab, and/or has not been administered asteroid, optionally dexamethasone, within a period of time followingadministration of the dose, which period of time is optionally at orabout 1, 2, 3, 4, 5 days or is optionally at or about 6, 7, 8, 9, 10, 11days or is optionally 1, 2, 3 or 4 weeks. In certain embodiments of anyof the provided methods, the subject is not administered an anti-IL-6 oranti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or hasnot been administered a steroid, optionally dexamethasone, followingadministration of the cell dose, prior to, or unless, the subjectexhibits a sign or symptom of a toxicity, optionally a neurotoxicity orCRS, and/or prior to, or unless, the subject exhibits a sign or symptomof a toxicity, optionally a neurotoxicity or CRS, other than a fever,optionally wherein the fever is not a sustained fever or the fever is orhas been reduced or reduced by more than 1° C. after treatment with anantipyretic. In certain embodiments of any of the provided methods, theadministration and any follow-up is carried out on an outpatient basisand/or without admitting the subject to a hospital and/or without anovernight stay at a hospital and/or without requiring admission to or anovernight stay at a hospital, optionally unless or until the subjectexhibits a sustained fever or a fever that is or has not been reduced ornot reduced by more than 1° C. after treatment with an antipyretic.

In some embodiments of any of the provided methods, the administrationis carried out on an outpatient basis and/or without requiring admissionto or an overnight stay at a hospital. In some embodiments of any of theprovided methods, if the subject, who is or has been treated on anoutpatient basis, exhibits a sustained fever or a fever that is or hasnot been reduced or not reduced by more than 1° C. after treatment withan antipyretic, the subject is admitted to the hospital or to anovernight stay at a hospital and/or is administered an agent ortreatment for the treatment or prevention or reduction or attenuation ofa neurotoxicity and/or a cytokine release syndrome or risk thereof.

In particular embodiments of any of the provided methods, the NHL isselected from the group consisting of aggressive NHL, diffuse large Bcell lymphoma (DLBCL), NOS (de novo and transformed from indolent),primary mediastinal large B cell lymphoma (PMBCL), mantle cell lymphoma(MCL), and/or follicular lymphoma (FL), optionally follicular lymphomaGrade 3B (FL3B). In certain embodiments of any of the provided methods,the NHL comprises diffuse large B cell lymphoma (DLBCL), primarymediastinal large B cell lymphoma (PMBCL), NOS (de novo or transformedfrom indolent lymphoma), or follicular lymphoma Grade 3B. In someexamples, the NHL includes DLBCL. In some embodiments of any of theprovided methods, the DLBCL is de novo or transformed from follicularlymphoma (FL) and/or does not comprise DLBCL transformed from MZL andCLL (Richter's).

In particular embodiments of any of the provided methods, the subject isor has been identified as having an Eastern Cooperative Oncology GroupPerformance Status (ECOG) status of 0, 1 or 2. In certain embodiments ofany of the provided methods, the subject is or has been identified ashaving an ECOG status of 0 or 1. In some embodiments of any of theprovided methods, at or immediately prior to the time of theadministration of the dose of cells the subject has relapsed followingremission after treatment with, or become refractory to, one or moreprior therapies for the NHL, optionally one, two or three priortherapies other than another dose of cells expressing the CAR.

In some embodiments of any of the provided methods, at or prior toadministration of the dose of cells, the subject is or has beenidentified as having a lymphoma associated with or involving centralnervous system (CNS) involvement. In some embodiments of any of theprovided methods, at least 70%, at least 80%, at least 90% or at least95% of subjects treated according to the method who, at or prior to theadministration of the dose of cells exhibited or were identified toexhibit a lymphoma with CNS involvement, achieved a resolution of theCNS disease.

In particular embodiments of any of the provided methods, at or prior tothe administration of the dose of cells: the subject is or has beenidentified as having a double/triple hit lymphoma (or high-grade B-celllymphoma, with MYC and BCL2 and/or BCL6 rearrangements with DLBCLhistology (double/triple hit)); the subject is or has been identified ashaving a chemorefractory lymphoma, optionally a chemorefractory DLBCL;the subject has not achieved complete remission (CR) in response to aprior therapy; and/or the subject has relapsed within 1 year or lessthan 1 year after receiving an autologous stem cell transplant (ASCT).

In some embodiments of any of the provided methods, the method includes,prior to administration of the dose of cells, identifying or selecting asubject for the administration of the dose of cells that has adouble/triple hit lymphoma (or high-grade B-cell lymphoma, with MYC andBCL2 and/or BCL6 rearrangements with DLBCL histology (double/triplehit)), a chemorefractory lymphoma, optionally a chemorefractory DLBCL,has not achieved complete remission (CR) in response to a prior therapyfor treating the malignancy, optionally the NHL; and/or has relapsedwithin 1 year or less than 1 year after receiving an autologous stemcell transplant (ASCT); and/or has a lymphoma associated with orinvolving central nervous system (CNS) involvement.

In some embodiments of any of the provided methods, the method furtherincludes administration of an additional therapeutic agent or therapy,optionally other than a cell therapy, optionally other than CAR⁺ T celltherapy. In some embodiments, the additional therapeutic agent ortherapy is for treating the NHL or malignancy and/or increases thepersistence, activity and/or efficacy of the dose of cells. In someembodiments, the additional therapeutic agent or therapy is administeredif the subject does not exhibit a response, optionally does not exhibita CR or OR, to the cell therapy within 1 month, within 2 months orwithin 3 months after administration of the dose of cells. In someembodiments, the additional therapeutic agent or therapy is administeredto a subject: that is or has been identified to have stable orprogressive disease (SD/PD) following treatment with a prior therapy,optionally a prior therapy with a chemotherapeutic agent, that is or hasbeen identified with an Eastern Cooperative Oncology Group PerformanceStatus (ECOG) status of 2, that is or has been identified as having atransformed follicular lymphoma (tFL) and/or that is or has beenidentified has having a DLBCL transformed from MZL and CLL. In someembodiments, prior to administration of the dose of cells or theadditional therapeutic agent or therapy, the method includes identifyingor selecting a subject for the administration of the dose of cells thathas stable or progressive disease (SD/PD) following treatment with aprior therapy, optionally a prior therapy with a chemotherapeutic agent,an Eastern Cooperative Oncology Group Performance Status (ECOG) statusof 2, a transformed follicular lymphoma (tFL) and/or a DLBCL transformedfrom MZL and CLL. In some of any of such embodiments, the additionaltherapeutic agent or therapy is administered prior to, with or at thesame time and/or subsequent to initiation of administration of the doseof cells.

In certain embodiments of any of the provided methods, the CAR comprisesan scFv specific for the antigen, a transmembrane domain, a cytoplasmicsignaling domain derived from a costimulatory molecule, which optionallyis a 4-1BB, and a cytoplasmic signaling domain derived from a primarysignaling ITAM-containing molecule, which optionally is a CD3zeta. Insome embodiments of any of the provided methods, the antigen is a B cellantigen, which optionally is CD19.

In particular embodiments of any of the provided methods, prior to theadministration, the subject has been preconditioned with alymphodepleting therapy comprising the administration of fludarabineand/or cyclophosphamide. Certain embodiments of any of the providedmethods further comprise, immediately prior to the administration,administering a lymphodepleting therapy to the subject comprising theadministration of fludarabine and/or cyclophosphamide. In someembodiments of any of the provided methods, the lymphodepleting therapycomprises administration of cyclophosphamide at about 200-400 mg/m²,optionally at or about 300 mg/m², inclusive, and/or fludarabine at about20-40 mg/m², optionally 30 mg/m², daily for 2-4 days, optionally for 3days. In particular embodiments of any of the provided methods, thelymphodepleting therapy comprises administration of cyclophosphamide ator about 300 mg/m² and fludarabine at about 30 mg/m²daily for 3 days.

In certain embodiments of any of the provided methods, theadministration of the cell dose and/or the lymphodepleting therapy iscarried out via outpatient delivery. In some embodiments of any of theprovided methods, the dose of cells is administered parenterally,optionally intravenously.

In particular embodiments of any of the provided methods: at least 40%or at least 50% of subjects treated according to the method achievecomplete remission (CR), exhibit progression-free survival (PFS) and/oroverall survival (OS) of greater than at or about 3 months, 6 months or12 months; on average, subjects treated according to the method exhibita median PFS or OS of greater than at or about 6 months, 12 months, or18 months; and/or the subject exhibits PFS or OS following therapy forat least at or about 6, 12, 18 or more months. In certain embodiments ofany of the provided methods, at or about 14 or 28 days after initiationof administration of the dose of cells, the number of CAR⁺ T cells,optionally CAR⁺ CD8⁺ T cells and/or CAR⁺ CD4⁺ T cells, detectable in theblood of the subject, or in a majority of subjects so treated by themethod, is greater than 1 cells per greater than 5 cells per μL orgreater than per 10 cells per μL.

In some embodiments of any of the provided methods, the T cells areprimary T cells obtained from a subject. In particular embodiments ofany of the provided methods, the T cells are autologous to the subject.In certain embodiments of any of the provided methods, the T cells areallogeneic to the subject. In some embodiments of any of the providedmethods, the T cells comprise CD4⁺ and CD8⁺ T cells administered as aplurality of compositions, said plurality of compositions comprisingadministration of a first composition comprising the CD4⁺ T cells or theCD8⁺ T cells and administration of a second composition comprising theother of the CD4⁺ T cells or the CD8⁺ T cells.

In particular embodiments of any of the provided methods, the firstcomposition and second composition are administered 0 to 12 hours apart,0 to 6 hours apart or 0 to 2 hours apart. In certain embodiments of anyof the provided methods, the first composition and second compositionare administered no more than 2 hours, no more than 1 hour, no more than30 minutes, no more than 15 minutes, no more than 10 minutes or no morethan 5 minutes apart. In some embodiments of any of the providedmethods, the first composition comprises the CD4⁺ T cell. In particularembodiments of any of the provided methods, the first compositioncomprises the CD8⁺ T cells. In certain embodiments of any of theprovided methods, the first composition is administered prior to thesecond composition.

In some embodiments of any of the provided methods, the dose of T cellsis administered to the subject as a single dose or is administered onlyone time within a period of two weeks, one month, three months, sixmonths, 1 year or more. In particular embodiments of any of the providedmethods, the dose of T cells is administered as a double dose comprisinga first dose of the T cells and a consecutive dose of the T cells,wherein one or both of the first dose and the second dose comprisesadministration of the plurality of compositions of T cells. In certainembodiments of any of the provided methods, the consecutive dose isadministered at a point in time that is at least or more than about 7days or 14 days after and less than about 28 days after initiation ofthe administration of the first dose of cells.

Provided herein is an article of manufacture comprising a cell therapycomprising a dose or composition of genetically engineered cellsexpressing a chimeric antigen receptor (CAR), and instructions foradministering the cell therapy, wherein the instructions specify: thedose of cells is to be administered to a subject having or identified tohave non-Hodgkin lymphoma (NHL), the NHL selected from diffuse large Bcell lymphoma (DLBCL), primary mediastinal large B cell lymphoma(PMBCL), NOS (de novo or transformed from indolent lymphoma), orfollicular lymphoma Grade 3B, wherein the subject is or has beenidentified as having an Eastern Cooperative Oncology Group PerformanceStatus (ECOG) status of 0 or 1; and the dose of T cells to beadministered comprises between at or about 5×10⁷ CAR-expressing T cellsand 1×10⁸ CAR-expressing T cells, inclusive. In some embodiments of anyof the provided articles of manufacture, the instructions specifyadministering the dose of T cells at a defined ratio of CD4⁺ cellsexpressing the CAR to CD8⁺ cells expressing the CAR and/or of CD4⁺ cellsto CD8⁺ cells, which ratio optionally is approximately 1:1 or is betweenapproximately 1:3 and approximately 3:1.

Provided herein is an article of manufacture comprising a cell therapycomprising a dose or composition of genetically engineered cellsexpressing a chimeric antigen receptor (CAR), and instructions foradministering the cell therapy, wherein the instructions specify: thedose of T cells is to be administered at a defined ratio of CD4⁺ cellsexpressing the CAR to CD8⁺ cells expressing the CAR and/or of CD4⁺ cellsto CD8⁺ cells, which ratio is approximately or is 1:1; and the dose ofcells is to be administered to a subject having or identified to havenon-Hodgkin lymphoma (NHL), the NHL selected from diffuse large B celllymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS(de novo or transformed from indolent lymphoma), or follicular lymphomaGrade 3B.

Provided herein is an article of manufacture comprising a cell therapycomprising a dose or composition of genetically engineered cellsexpressing a chimeric antigen receptor (CAR), and instructions foradministering the cell therapy, wherein the instructions specify: thedose of cells is to be administered to a subject having or identified tohave non-Hodgkin lymphoma (NHL), optionally an NHL selected fromaggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo andtransformed from indolent), primary mediastinal large B cell lymphoma(PMBCL), mantle cell lymphoma (MCL), and/or follicular lymphoma (FL),optionally follicular lymphoma Grade 3B (FL3B), the dose of T cells tobe administered comprises between at or about 5×10⁷ CAR-expressing Tcells and 1×10⁸ CAR-expressing T cells, inclusive; and the dose of Tcells is to be administered at a defined ratio of CD4⁺ cells expressingthe CAR to CD8⁺ cells expressing the CAR and/or of CD4⁺ cells to CD8⁺cells, which ratio is approximately or is 1:1.

In some embodiments of any of the provided articles of manufacture, theinstructions further specify the dose of cells is to be administered toa subject that is or has been identified as having an EasternCooperative Oncology Group Performance Status (ECOG) status of 0, 1 or2, optionally an ECOG status of 0 or 1. In certain embodiments of any ofthe provided articles of manufacture, the instructions specify that theadministration is in a subject that has not received, immediately priorto the administration of the dose of cells or within or about 1 month ofthe dose of cells, an agent or treatment capable of treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity. In some embodiments of any of the providedarticles of manufacture, the agent is or comprises an anti-IL-6 oranti-IL-6R antibody, optionally tocilizumab or siltuximab, and/or asteroid, optionally dexamethasone. In particular embodiments of any ofthe provided articles of manufacture, the instructions specify the doseof cells is not for administration in a subject having DLBCL transformedfrom MZL and CLL (Richter's) and/or is for a subject having a DLBCL thatis de novo or transformed from indolent disease. In some embodiments ofany of the provided articles of manufacture, the instructions specifythe subject does not have a DLBCL transformed from MZL and CLL(Richter's).

In some embodiments of any of the provided articles of manufacture, theinstructions specify the administration of the cell therapy is for asubject that is or has been identified as having a double/triple hitlymphoma (or high-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6rearrangements with DLBCL histology (double/triple hit)), is or has beenidentified as having a chemorefractory lymphoma, optionally achemorefractory DLBCL; and/or that has not achieved complete remission(CR) in response to a prior therapy. In certain embodiments of any ofthe provided articles of manufacture, the CAR comprises an scFv specificfor the antigen, a transmembrane domain, a cytoplasmic signaling domainderived from a costimulatory molecule, which optionally is a 4-1BB, anda cytoplasmic signaling domain derived from a primary signalingITAM-containing molecule, which optionally is a CD3zeta. In certainembodiments of any of the provided articles of manufacture, the antigenis a B cell antigen, which optionally is CD19.

Particular embodiments of any of the provided articles of manufacturefurther comprise instructions for use with, after or in connection witha lymphodepleting therapy, the lymphodepleting therapy optionallycomprising fludarabine and/or cyclophosphamide. In certain embodimentsof any of the provided articles of manufacture, the lymphodepletingtherapy comprises administration of cyclophosphamide at about 200-400mg/m², optionally at or about 300 mg/m², inclusive, and/or fludarabineat about 20-40 mg/m², optionally 30 mg/m², daily for 2-4 days,optionally for 3 days. In particular embodiments of any of the providedarticles of manufacture, the lymphodepleting therapy comprisesadministration of cyclophosphamide at or about 300 mg/m² and fludarabineat about 30 mg/m²daily for 3 days.

In some embodiments of any of the provided articles of manufacture, theinstructions further specify the administration of the cell therapy isto be or may be administered to the subject on an outpatient settingand/or without admission of the subject to the hospital overnight or forone or more consecutive days and/or is without admission of the subjectto the hospital for one or more days. In certain embodiments of any ofthe provided articles of manufacture, the instructions further specifythe cell therapy is for parenteral administration, optionallyintravenous administration. In particular embodiments of any of theprovided articles of manufacture, the cell therapy comprises primary Tcells obtained from a subject. In some embodiments of any of theprovided articles of manufacture, the T cells are autologous to thesubject. In certain embodiments of any of the provided articles ofmanufacture, the T cells are allogeneic to the subject.

In particular embodiments of any of the provided articles ofmanufacture, the article of manufacture comprises a plurality ofcompositions of the cell therapy, the plurality of compositionscomprising a first composition of genetically engineered cellscomprising CD4⁺ T cells or CD8⁺ T cells, wherein the instructionsspecify the first composition is for use in with a second compositioncomprising the other of the CD4⁺ T cells or the CD8⁺ T cells, optionallywherein the cells of the first composition and cells of the samecomposition are from the same subject.

In some embodiments of any of the provided articles of manufacture, theinstructions specify the first composition and second composition are tobe administered at a defined ratio of CD4⁺ cells expressing therecombinant receptor to CD8⁺ cells expressing the recombinant receptorand/or of CD4⁺ cells to CD8⁺ cells, which ratio optionally isapproximately 1:1 or is between approximately 1:3 and approximately 3:1.In certain embodiments of any of the provided articles of manufacture,the defined ratio is or is approximately 1:1. In particular embodimentsof any of the provided articles of manufacture, the composition furthercomprises a cryoprotectant and/or the article further includesinstructions for thawing the composition prior to administration to thesubject.

In some embodiments of any of the provided articles of manufacture, theinstructions specify administering the composition comprising the CD4⁺ Tcells and the composition comprising the CD8⁺ T cells 0 to 12 hoursapart, 0 to 6 hours apart or 0 to 2 hours apart. In certain embodimentsof any of the provided articles of manufacture, the instructions specifyadministering the composition comprising the CD4⁺ T cells and thecomposition comprising the CD8⁺ T cells no more than 2 hours, no morethan 1 hour, no more than 30 minutes, no more than 15 minutes, no morethan 10 minutes or no more than 5 minutes apart. In particularembodiments of any of the provided articles of manufacture, theinstructions specify administering the composition comprising the CD4⁺ Tcells prior to administering the composition comprising the CD8⁺ cells.In some embodiments of any of the provided articles of manufacture, theinstructions specify administering the composition comprising the CD8⁺ Tcells prior to administering the composition comprising the CD4⁺ cells.

Provided herein are articles of manufacture comprising one or morereagent capable of detecting one or more analytes, and instructions forusing the reagent to assay a biological sample from a subject that is acandidate for treatment, optionally with a cell therapy, said celltherapy optionally comprising a dose or composition of geneticallyengineered cells expressing a recombinant receptor, wherein the one ormore analytes is selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8,IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin,G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, and D-dimer (fibrindegradation product).

Particular embodiments of any of the provided articles of manufacturefurther comprise the cell therapy and/or further comprising instructionsfor use with, prior to and/or in connection with treatment with the celltherapy. Certain embodiments of any of the provided articles ofmanufacture further comprise one or more agents or treatments fortreating, preventing, delaying, reducing or attenuating the developmentor risk of development of a toxicity and/or instructions for theadministration of one or more agents or treatments for treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity in the subject.

In some embodiments of any of the provided articles of manufacture, theinstructions further specify, if the level, amount or concentration ofthe analyte in the sample is at or above a threshold level for theanalyte: administering to the subject an agent or other treatmentcapable of treating, preventing, delaying, reducing or attenuating thedevelopment or risk of development of a toxicity (i) prior to, (ii)within one, two, or three days of, (iii) concurrently with and/or (iv)at first fever following, the initiation of administration of the celltherapy to the subject; and/or administering to the subject the celltherapy at a reduced dose or at a dose that is not associated with riskof developing toxicity or severe toxicity, or is not associated with arisk of developing a toxicity or severe toxicity in a majority ofsubjects, and/or a majority of subjects having a disease or conditionthat the subject has or is suspected of having, following administrationof the cell therapy; and/or administering to the subject the celltherapy in an in-patient setting and/or with admission to the hospitalfor one or more days, optionally wherein the cell therapy is otherwiseto be administered to subjects on an outpatient basis or withoutadmission to the hospital for one or more days.

In particular embodiments of any of the provided articles ofmanufacture, the instructions further specify, if the level, amount orconcentration of the analyte is below a threshold level for the analyte,administering to the subject the cell therapy, optionally at anon-reduced dose, optionally on an outpatient basis or without admissionto the hospital for one or more days.

In certain embodiments of any of the provided articles of manufacture,the instructions further specify administering the cell therapy to thesubject and wherein the instructions further specify, if the level,amount or concentration of the analyte, is below a threshold level: theadministration of the cell therapy does not comprise administering,prior to or concurrently with administering the cell therapy and/orprior to the development of a sign of symptom of a toxicity other thanfever, an agent or treatment capable of treating, preventing, delaying,or attenuating the development of the toxicity; and/or theadministration of the cell therapy is to be or may be administered tothe subject on an outpatient setting and/or without admission of thesubject to the hospital overnight or for one or more consecutive daysand/or is without admission of the subject to the hospital for one ormore days.

In some embodiments of any of the provided articles of manufacture, thethreshold level is within 25%, within 20%, within 15%, within 10% orwithin 5% of the average level, amount or concentration, and/or iswithin a standard deviation of the average level, amount orconcentration, of the analyte in a biological sample obtained from agroup of subjects prior to receiving a recombinant receptor-expressingtherapeutic cell composition, wherein each of the subjects of the groupwent on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition. Provided herein is an article ofmanufacture comprising a cell therapy, said cell therapy optionallycomprising a dose or composition of genetically engineered cellsexpressing a recombinant receptor, and instructions for administeringthe cell therapy following or based on the results of an assessment, ina biological sample of the level, or amount or concentration of one ormore analyte in a biological sample, said biological sample obtainedfrom the subject prior to administering the cell therapy and/or saidbiological sample not comprising the recombinant receptor and/or saidengineered cells, wherein the one or more analytes is selected from LDH,ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha,IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta,IP-10, perforin, and D-dimer (fibrin degradation product).

In particular embodiments of any of the provided articles ofmanufacture, said assessment comprises detection which optionallycomprises contacting a reagent capable of directly or indirectlydetecting the analyte with the biological sample and determining thelevel, amount or concentration of the analyte in the biological sample.Certain embodiments of any of the provided articles of manufacturefurther comprise the reagent and/or further comprising instructions foruse with, prior to and/or in connection with the reagent for detectingthe analyte. Some embodiments of any of the provided articles ofmanufacture further comprise one or more agents or treatments fortreating, preventing, delaying, reducing or attenuating the developmentor a risk of development of a toxicity and/or instructions for theadministration of one or more agents or treatments for treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity in the subject.

In particular embodiments of any of the provided articles ofmanufacture, the instructions for administering the cell therapyspecify, if the level, amount or concentration of the analyte in thesample, is at or above a threshold level: administering to the subjectan agent or other treatment capable of treating, preventing, delaying,reducing or attenuating the development or risk of development of atoxicity (i) prior to, (ii) within one, two, or three days of, (iii)concurrently with and/or (iv) at first fever following, the initiationof administration of administration of the therapeutic cell compositionor the genetically engineered cells; and/or administering to the subjectthe cell therapy at a reduced dose or at a dose that is not associatedwith risk of developing toxicity or severe toxicity, or is notassociated with a risk of developing a toxicity or severe toxicity in amajority of subjects, and/or a majority of subjects having a disease orcondition that the subject has or is suspected of having, followingadministration of the cell therapy; and/or administering to the subjectthe cell therapy in an in-patient setting and/or with admission to thehospital for one or more days, optionally wherein the cell therapy isotherwise to be administered to subjects on an outpatient basis orwithout admission to the hospital for one or more days.

In certain embodiments of any of the provided articles of manufacture,the instructions for administering the cell therapy specify, if thelevel, amount or concentration of the analyte in the sample, is below athreshold level, administering to the subject the cell therapy,optionally at a non-reduced dose, optionally on an outpatient basis orwithout admission to the hospital for one or more days. In someembodiments of any of the provided articles of manufacture, theinstructions further specify administering the cell therapy to thesubject and wherein the instructions further specify, if the level,amount or concentration of the analyte is below a threshold level: notadministering, prior to or concurrently with administering the celltherapy and/or prior to the development of a sign or symptom of atoxicity other than fever, an agent or treatment capable of treating,preventing, delaying, or attenuating the development of the toxicity;and/or the administration of the cell therapy is to be or may beadministered to the subject on an outpatient setting and/or withoutadmission of the subject to the hospital overnight or for one or moreconsecutive days and/or is without admission of the subject to thehospital for one or more days.

In particular embodiments of any of the provided articles ofmanufacture, the threshold level is within 25%, within 20%, within 15%,within 10% or within 5% of the average level, amount or concentration,and/or is within a standard deviation of the average level, amount orconcentration, of the analyte in a biological sample obtained from agroup of subjects prior to receiving a recombinant receptor-expressingtherapeutic cell composition, wherein each of the subjects of the groupwent on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

Provided herein are articles of manufacture comprising an agent capableof treating, preventing, delaying, reducing or attenuating thedevelopment or risk of development of a toxicity, and instructions foradministering the agent following or based on the results of anassessment in a biological sample of the level, amount or concentrationof one or more analytes in a biological sample, wherein the one or moreanalytes is selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10,IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF,IL-1Ralpha, IL-1Rbeta, IP-10, perforin, and D-dimer (fibrin degradationproduct). In certain embodiments of any of the provided articles ofmanufacture, said assessment comprises detection which optionallycomprises contacting a reagent capable of directly or indirectlydetecting the analyte with the biological sample and determining thelevel, amount or concentration of the analyte in the biological sample.

In some embodiments of any of the provided articles of manufacture, theinstructions specify that the agent is to be administered i) prior to,(ii) within one, two, or three days of, (iii) concurrently with and/or(iv) at first fever following, the initiation of administration of thecell therapy to the subject and/or further comprises instructions foruse with, prior to and/or in connection with treatment with the celltherapy. In particular embodiments of any of the provided articles ofmanufacture, said biological sample is obtained from the subject priorto administering the agent or cell therapy. In certain embodiments ofany of the provided articles of manufacture, the reagent is a bindingmolecule that specifically binds to the marker or cells of the myeloidcell population. In some embodiments of any of the provided articles ofmanufacture, the reagent is an antibody or an antigen-binding fragmentthereof. In particular embodiments of any of the provided articles ofmanufacture, the biological sample is or is obtained from a blood,plasma or serum sample. In certain embodiments of any of the providedarticles of manufacture, comprising the reagent for detecting theanalyte and/or further comprising instructions for use with, prior toand/or in connection with the reagent for detecting the analyte. Someembodiments of any of the provided articles of manufacture furthercomprise the cell therapy and/or further comprising instructions for usewith, prior to and/or in connection with treatment with the celltherapy.

In particular embodiments of any of the provided articles ofmanufacture, the instructions for administering the agent specify, ifthe level, amount or concentration of the analyte in the sample, is ator above a threshold level administering to the subject the agent. Incertain embodiments of any of the provided articles of manufacture, theinstruction further specify administering a cell therapy to the subject,wherein administration of the agent is to be carried out (i) prior to,(ii) within one, two, or three days of, (iii) concurrently with and/or(iv) at first fever following, the initiation of administration of thecell therapy to the subject. In some embodiments of any of the providedarticles of manufacture, the instructions for administering the agentspecify, if the level, amount or concentration is below the thresholdlevel administering to the subject the cell therapy, optionally whereinthe instructions specify the cell therapy is to be or may beadministered to the subject on an outpatient setting and/or withoutadmission of the subject to the hospital overnight or for one or moreconsecutive days and/or is without admission of the subject to thehospital for one or more days.

In particular embodiments of any of the provided articles ofmanufacture, the threshold level is within 25%, within 20%, within 15%,within 10% or within 5% of the average level, amount or concentration,and/or is within a standard deviation of the average level, amount orconcentration, of the analyte in a biological sample obtained from agroup of subjects prior to receiving a recombinant receptor-expressingtherapeutic cell composition, wherein each of the subjects of the groupwent on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition. In certain embodiments of any ofthe provided articles of manufacture, assaying or assessing cells forthe analyte is by an immunoassay.

In some embodiments of any of the provided articles of manufacture, thetoxicity comprises neurotoxicity or cytokine release syndrome (CRS),optionally grade 1 or higher neurotoxicity or CRS. In particularembodiments of any of the provided articles of manufacture: the toxicitycomprises severe neurotoxicity and/or comprises a grade 2 or higherneurotoxicity, a grade 3 or higher neurotoxicity, at least prolongedgrade 3 neurotoxicity or is at or above grade 4 or grade 5neurotoxicity; and/or the toxicity comprises severe CRS and/or comprisesgrade 2 or higher or grade 3 or higher CRS. In certain embodiments ofany of the provided articles of manufacture, the toxicity is associatedwith cerebral edema.

In some embodiments of any of the provided articles of manufacture, theagent or other treatment is or comprises one or more of a steroid; anantagonist or inhibitor of a cytokine receptor or cytokine selected fromamong IL-10, IL-10R, IL-6, IL-6 receptor, IFNγ, IFNGR, IL-2, IL-2R/CD25,MCP-1, CCR2, CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1, andIL-1Ralpha/IL-1beta; or an agent capable of preventing, blocking orreducing microglial cell activity or function. In particular embodimentsof any of the provided articles of manufacture, the antagonist orinhibitor is or comprises an agent selected from among an antibody orantigen-binding fragment, a small molecule, a protein or peptide and anucleic acid. In certain embodiments of any of the provided articles ofmanufacture, the agent or other treatment is an anti-IL-6 antibody or ananti-IL6 receptor antibody.

In some embodiments of any of the provided articles of manufacture, theagent or other treatment is or comprises an agent selected from amongtocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab (CDP6038),elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634,ARGX-109, FE301 and FM101. In particular embodiments of any of theprovided articles of manufacture, the agent or other treatment is orcomprises tocilizumab. In certain embodiments of any of the providedarticles of manufacture, the agent or other treatment is or comprisessiltuximab. In some embodiments of any of the provided articles ofmanufacture, the steroid is or comprises dexamethasone.

In particular embodiments of any of the provided articles ofmanufacture, the agent capable of preventing, blocking or reducingmicroglial cell activity or function is selected from ananti-inflammatory agent, an inhibitor of NADPH oxidase (NOX2), a calciumchannel blocker, a sodium channel blocker, inhibits GM-CSF, inhibitsCSF1R, specifically binds CSF-1, specifically binds IL-34, inhibits theactivation of nuclear factor kappa B (NF-κB), activates a CB₂ receptorand/or is a CB₂ agonist, a phosphodiesterase inhibitor, inhibitsmicroRNA-155 (miR-155) or upregulates microRNA-124 (miR-124). In certainembodiments of any of the provided articles of manufacture, the agentcapable of preventing, blocking or reducing microglial cell activationor function is a small molecule, peptide, protein, antibody orantigen-binding fragment thereof, an antibody mimetic, an aptamer, or anucleic acid molecule.

In some embodiments of any of the provided articles of manufacture, theagent is selected from minocycline, naloxone, nimodipine, Riluzole,MOR103, lenalidomide, a cannabinoid (optionally WIN55 or 212-2),intravenous immunoglobulin (IVIg), ibudilast, anti-miR-155 lockednucleic acid (LNA), MCS110, PLX-3397, PLX647, PLX108-D1, PLX7486,JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945, emactuzumab, IMC-CS4, FPA008,LY-3022855, AMG-820 and TG-3003. In particular embodiments of any of theprovided articles of manufacture, the agent is an inhibitor of colonystimulating factor 1 receptor (CSF1R). In certain embodiments of any ofthe provided articles of manufacture, the inhibitor is selected from:PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141,ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945 or a pharmaceutical salt or prodrugthereof; emactuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003or is an antigen-binding fragment thereof; or a combination of any ofthe foregoing. In some embodiments of any of the provided articles ofmanufacture, the inhibitor is PLX-3397.

In certain embodiments of any of the provided articles of manufacture,the disease or condition is a cancer. In particular embodiments of anyof the provided articles of manufacture, the disease or condition is amyeloma, leukemia or lymphoma. In some embodiments of any of theprovided articles of manufacture, the disease or condition is a B cellmalignancy and/or is acute lymphoblastic leukemia (ALL), adult ALL,chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), andDiffuse Large B-Cell Lymphoma (DLBCL).

In certain embodiments of any of the provided articles of manufacture,the antigen is ROR1, B cell maturation antigen (BCMA), carbonicanhydrase 9 (CAIX), tEGFR, Her2/neu (receptor tyrosine kinase erbB2),L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surfaceantigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR,epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40),EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate bindingprotein (FBP), FCRLS, FCRHS, fetal acetylcholine receptor, GD2, GD3,HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor(kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM),Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentiallyexpressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AIMAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8,avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2Dligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin,murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100,oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA),Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), acyclin, cyclin A2, CCL-1, CD138, G Protein Coupled Receptor 5D (GPCRSD),or a pathogen-specific antigen.

In particular embodiments of any of the provided articles ofmanufacture, the recombinant receptor is a T cell receptor or afunctional non-T cell receptor. In some embodiments of any of theprovided articles of manufacture, the recombinant receptor is a chimericantigen receptor (CAR). In certain embodiments of any of the providedarticles of manufacture, the CAR comprises an extracellularantigen-recognition domain that specifically binds to the antigen and anintracellular signaling domain comprising an ITAM, wherein optionally,the intracellular signaling domain comprises an intracellular domain ofa CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises acostimulatory signaling region, which optionally comprises a signalingdomain of CD28 or 4-1BB.

In particular embodiments of any of the provided articles ofmanufacture, the engineered cells comprise T cells, optionally CD4⁺and/or CD8⁺. In some embodiments of any of the provided articles ofmanufacture, the T cells are primary T cells obtained from a subject. Incertain embodiments of any of the provided articles of manufacture, thedose that is not associated with risk of developing toxicity or severetoxicity is or comprises less than or less than about 5×10⁷ totalrecombinant receptor-expressing cells, optionally CAR⁺ cells, total Tcells, or total peripheral blood mononuclear cells (PBMCs), such as lessthan or less than about 2.5×10⁷, less than or less than about 1.0×10⁷,less than or less than about 5.0×10⁶, less than or less than about1.0×10⁶, less than or less than about 5.0×10⁵, or less than or less thanabout 1×10⁵ total recombinant receptor-expressing cells, optionally CAR⁺cells, total T cells, or total peripheral blood mononuclear cells(PBMCs). In particular embodiments of any of the provided articles ofmanufacture, the dose that is not associated with risk of developingtoxicity or severe toxicity is or comprises from or from about 1×10⁵ to5×10⁷ total recombinant receptor-expressing cells, optionally CAR⁺cells, total T cells, or total peripheral blood mononuclear cells(PBMCs), such as 1×10⁵ to 2.5×10⁷, 1×10⁵ to 1.0×10⁷, 1×10⁵ to 5.0×10⁶,1×10⁵ to 1.0×10⁶, 1.0×10⁵ to 5.0×10⁵, 5.0×10⁵ to 5×10⁷, 5×10⁵ to2.5×10⁷, 5×10⁵ to 1.0×10⁷, 5×10⁵ to 5.0×10⁶, 5×10⁵ to 1.0×10⁶, 1.0×10⁶to 5×10⁷, 1×10⁶ to 2.5×10⁷, 1×10⁶ to 1.0×10⁷, 1×10⁶ to 5.0×10⁶, 5.0×10⁶to 5×10⁷, 5×10⁶ to 2.5×10⁷, 5×10⁶ to 1.0×10⁷, 1.0×10⁷ to 5×10⁷, 1×10⁷ to2.5×10⁷ or 2.5×10⁷ to 5×10⁷ total recombinant receptor-expressing cells,optionally CAR⁺ cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs).

In certain embodiments of any of the provided articles of manufacture,the reagent is detectably labeled, optionally fluorescently labeled. Insome embodiments of any of the provided articles of manufacture, the oneor more analyte is LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-alpha,IFN-alpha2, MCP-1 and MCP-1beta. In particular embodiments of any of theprovided articles of manufacture, the one or more analyte is orcomprises LDH.

Provided herein are methods of selecting a subject for treatment, themethod comprising: (a) contacting a biological sample with one or morereagent capable of detecting or that is specific for one or moreanalyte, wherein the one or more analyte is selected from LDH, ferritin,CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1,MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, andD-dimer (fibrin degradation product), wherein: the biological sample isfrom a subject that is a candidate for treatment with a cell therapy,said cell therapy optionally comprising a dose or composition ofgenetically engineered cells expressing a recombinant receptor; and thebiological sample is obtained from the subject prior to administeringthe cell therapy and/or said biological sample does not comprise therecombinant receptor and/or said engineered cells; and (b) selecting asubject in which either: (i) the level, amount or concentration of theanalyte in the samples at or above a threshold level, therebyidentifying a subject that is at risk for developing a toxicity to thecell therapy; or (ii) the level, amount or concentration of the analyteis below a threshold level.

In certain embodiments of any of the provided methods: (a) a subject in(i) is selected for administering to the subject (1) an agent or othertreatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity and (2)the cell therapy, wherein administration of the agent is to beadministered (i) prior to, (ii) within one, two, or three days of, (iii)concurrently with and/or (iv) at first fever following, the initiationof administration of the cell therapy to the subject; and/or (c) asubject in (i) is selected for administering to the subject a celltherapy at a reduced dose or at a dose that is not associated with riskof developing toxicity or severe toxicity, or is not associated with arisk of developing a toxicity or severe toxicity in a majority ofsubjects, and/or a majority of subjects having a disease or conditionthat the subject has or is suspected of having, following administrationof the cell therapy; and/or (b) a subject in (i) is selected foradministering to the subject a cell therapy in an in-patient settingand/or with admission to the hospital for one or more days, optionallywherein the cell therapy is otherwise to be administered to subjects onan outpatient basis or without admission to the hospital for one or moredays.

In some embodiments of any of the provided methods, a subject in (i) isselected, and the method further comprises: (a) administering to thesubject (1) an agent or other treatment capable of treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity and (2) the cell therapy, wherein administration of theagent is carried out (i) prior to, (ii) within one, two, or three daysof, (iii) concurrently with and/or (iv) at first fever following, theinitiation of administration of the cell therapy to the subject; and/or(b) administering to the subject a cell therapy at a reduced dose or ata dose that is not associated with risk of developing toxicity or severetoxicity, or is not associated with a risk of developing a toxicity orsevere toxicity in a majority of subjects, and/or a majority of subjectshaving a disease or condition that the subject has or is suspected ofhaving, following administration of the cell therapy; and/or (c)administering to the subject a cell therapy or a dose of geneticallyengineered cells of a cell therapy that is not associated with risk ofdeveloping toxicity or severe toxicity, or is not associated with a riskof developing a toxicity or severe toxicity in a majority of subjects,and/or a majority of subjects having a disease or condition that thesubject has or is suspected of having, following administration of thecell therapy; and/or (d) administering to the subject a cell therapy inan in-patient setting and/or with admission to the hospital for one ormore days, optionally wherein the cell therapy is otherwise to beadministered to subjects on an outpatient basis or without admission tothe hospital for one or more days.

In particular embodiments of any of the provided methods: (a) a subjectin (ii) is selected for administering to the subject a cell therapy,optionally at a non-reduced dose, optionally on an outpatient basis orwithout admission to the hospital for one or more days; (b) a subject in(ii) is selected for administering to the subject a cell therapy,wherein the cell therapy does not comprise administering, prior to orconcurrently with administering the cell therapy and/or prior to thedevelopment of a sign or symptom of a toxicity other than fever, anagent or treatment capable of treating, preventing, delaying, orattenuating the development of the toxicity; and/or a subject in (ii) isselected for administering a cell therapy on an outpatient settingand/or without admission of the subject to the hospital overnight or forone or more consecutive days and/or is without admission of the subjectto the hospital for one or more days.

In certain embodiments of any of the provided methods, a subject in (ii)is selected, and the method further comprises administering to thesubject the cell therapy, optionally at a non-reduced dose, optionallyon an outpatient basis or without admission to the hospital for one ormore days. In some embodiments of any of the provided methods, a subjectin (ii) is selected, and the method further comprises administering tothe subject the cell therapy, wherein: the administration of the celltherapy does not comprise administering, prior to or concurrently withadministering the cell therapy and/or prior to the development of a signor symptom of a toxicity other than fever, an agent or treatment capableof treating, preventing, delaying, or attenuating the development of thetoxicity; and/or the administration of the cell therapy is to be or maybe administered to the subject on an outpatient setting and/or withoutadmission of the subject to the hospital overnight or for one or moreconsecutive days and/or is without admission of the subject to thehospital for one or more days.

Provided herein is a method of treatment, comprising: (a) assaying abiological sample for the level, amount or concentration of one or moreanalyte, wherein the biological sample is from a subject that is acandidate for treatment, optionally with a cell therapy, said celltherapy optionally comprising a dose or composition of geneticallyengineered cells expressing a recombinant receptor for treating adisease or condition, wherein the one or more analyte is selected fromLDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha,IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta,IP-10, perforin, and D-dimer (fibrin degradation product); and (b)following or based on the results of the assay, administering to thesubject the cell therapy, and, optionally, an agent or other treatmentcapable of treating, preventing, delaying, reducing or attenuating thedevelopment or risk of development of a toxicity.

Provided herein is a method of treatment, comprising, following or basedon the results of an assay, of a biological sample from a subject, forthe level, amount or concentration of one or more analyte, administeringto the subject (i) a cell therapy, optionally comprising a dose orcomposition of genetically engineered expressing a recombinant receptorfor treating a disease or condition, and, optionally, (ii) an agent orother treatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity,wherein: the biological sample is obtained from the subject prior toadministering the cell therapy; and the one or more analyte is selectedfrom LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16,TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha,IL-1Rbeta, IP-10, perforin, and D-dimer (fibrin degradation product).

In particular embodiments of any of the provided methods, said assayingcomprises detection which optionally comprises contacting a reagentcapable of directly or indirectly detecting the analyte with thebiological sample and determining the level, amount or concentration ofthe analyte in the biological sample. In certain embodiments of any ofthe provided methods, if the level, amount or concentration of theanalyte in the sample, is at or above a threshold level: administeringto the subject the agent or other treatment capable of treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity (i) prior to, (ii) within one, two, or threedays of, (iii) concurrently with and/or (iv) at first fever following,the initiation of administration of the cell therapy to the subject;and/or administering to the subject the cell therapy at a reduced doseor at a dose that is not associated with risk of developing toxicity orsevere toxicity, or is not associated with a risk of developing atoxicity or severe toxicity in a majority of subjects, and/or a majorityof subjects having a disease or condition that the subject has or issuspected of having, following administration of the cell therapy;and/or administering to the subject the cell therapy in an in-patientsetting and/or with admission to the hospital for one or more days,optionally wherein the cell therapy is otherwise to be administered tosubjects on an outpatient basis or without admission to the hospital forone or more days.

In some embodiments of any of the provided methods, if the level, amountor concentration of the analyte, is at or above a threshold level: theadministration of the cell therapy does not comprise administering,prior to or concurrently with administering the cell therapy and/orprior to the development of a sign or symptom of a toxicity other thanfever, an agent or treatment capable of treating, preventing, delaying,or attenuating the development of the toxicity; and/or theadministration of the cell therapy is to be or may be administered tothe subject on an outpatient setting and/or without admission of thesubject to the hospital overnight or for one or more consecutive daysand/or is without admission of the subject to the hospital for one ormore days.

In particular embodiments of any of the provided methods, administering,to a subject, an agent or other treatment capable of treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity, wherein: the subject is a candidate fortreatment optionally with a cell therapy, said cell therapy optionallycomprising a dose or composition of genetically engineered cellsexpressing a recombinant receptor for treating a disease or condition;and the subject has been identified as at risk for developing a toxicityfollowing or based on the results of an assay, of a biological samplefrom a subject, for the level, amount or concentration of one or moreanalyte, said biological sample obtained from the subject prior toadministering the cell therapy and/or said biological sample notcomprising the recombinant receptor and/or said engineered cells,wherein the one or more analyte is selected from LDH, ferritin, CRP,IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1,MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, andD-dimer (fibrin degradation product).

In certain embodiments of any of the provided methods, said assaycomprises detection which optionally comprises contacting a reagentcapable of directly or indirectly detecting the analye with thebiological sample and determining the level, amount or concentration ofthe analyte in the biological sample. In some embodiments of any of theprovided methods, the agent is administered to the subject if the level,amount or concentration of the analyte in the sample is at or above athreshold level.

In particular embodiments of any of the provided methods, the agent isadministered (i) prior to, (ii) within one, two, or three days of, (iii)concurrently with and/or (iv) at first fever following, the initiationof administration of the cell therapy to the subject. In certainembodiments of any of the provided methods, the threshold level iswithin 25%, within 20%, within 15%, within 10% or within 5% of theaverage percent or number, and/or is within a standard deviation of theaverage percent or number, of cells surface positive for the myeloidmarker in a biological sample obtained from a group of subjects prior toreceiving a recombinant receptor-expressing therapeutic cellcomposition, wherein each of the subjects of the group went on todevelop a toxicity after receiving a recombinant-receptor-expressingtherapeutic cell composition for treating the same disease or condition.

In particular embodiments of any of the provided methods, the thresholdlevel is within 25%, within 20%, within 15%, within 10% or within 5% ofthe average level, amount or concentration, and/or is within a standarddeviation of the average level, amount or concentration, of the analytein a biological sample obtained from a group of subjects prior toreceiving a recombinant receptor-expressing therapeutic cellcomposition, wherein each of the subjects of the group went on todevelop a toxicity after receiving a recombinant-receptor-expressingtherapeutic cell composition for treating the same disease or condition.In certain embodiments of any of the provided methods, the reagent is abinding molecule that specifically binds to the marker or cells of themyeloid cell population. In some embodiments of any of the providedmethods, the reagent is an antibody or an antigen-binding fragmentthereof. In particular embodiments of any of the provided methods, thebiological sample is or is obtained from a blood, plasma or serumsample. In certain embodiments of any of the provided methods, assayingor assessing cells the analyte comprises an immunoassay.

In some embodiments of any of the provided methods, the toxicitycomprises neurotoxicity or cytokine release syndrome (CRS), optionallygrade 1 or higher neurotoxicity or CRS. In particular embodiments of anyof the provided methods: the toxicity comprises severe neurotoxicityand/or comprises a grade 2 or higher neurotoxicity, a grade 3 or higherneurotoxicity, at least prolonged grade 3 neurotoxicity or is at orabove grade 4 or grade 5 neurotoxicity; and/or the toxicity comprisessevere CRS and/or comprises grade 2 or higher or grade 3 or higher CRS.In certain embodiments of any of the provided methods, the toxicity isassociated with cerebral edema.

In some embodiments of any of the provided methods, the agent or othertreatment is or comprises one or more of a steroid; an antagonist orinhibitor of a cytokine receptor or cytokine selected from among IL-10,IL-10R, IL-6, IL-6 receptor, IFNγ, IFNGR, IL-2, IL-2R/CD25, MCP-1, CCR2,CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1, and IL-1Ralpha/IL-1beta; or anagent capable of preventing, blocking or reducing microglial cellactivity or function. In particular embodiments of any of the providedmethods, the antagonist or inhibitor is or comprises an agent selectedfrom among an antibody or antigen-binding fragment, a small molecule, aprotein or peptide and a nucleic acid.

In certain embodiments of any of the provided methods, the agent orother treatment is an anti-IL-6 antibody or an anti-IL6 receptorantibody. In some embodiments of any of the provided methods, the agentor other treatment is or comprises an agent selected from amongtocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab (CDP6038),elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634,ARGX-109, FE301 and FM101. In particular embodiments of any of theprovided methods, the agent or other treatment is or comprisestocilizumab. In certain embodiments of any of the provided methods, theagent or other treatment is or comprises siltuximab.

In some embodiments of any of the provided methods, the steroid is orcomprises dexamethasone. In particular embodiments of any of theprovided methods, the agent capable of preventing, blocking or reducingmicroglial cell activity or function is selected from ananti-inflammatory agent, an inhibitor of NADPH oxidase (NOX2), a calciumchannel blocker, a sodium channel blocker, inhibits GM-CSF, inhibitsCSF1R, specifically binds CSF-1, specifically binds IL-34, inhibits theactivation of nuclear factor kappa B (NF-κB), activates a CB₂ receptorand/or is a CB₂ agonist, a phosphodiesterase inhibitor, inhibitsmicroRNA-155 (miR-155) or upregulates microRNA-124 (miR-124).

In certain embodiments of any of the provided methods, the agent capableof preventing, blocking or reducing microglial cell activation orfunction is a small molecule, peptide, protein, antibody orantigen-binding fragment thereof, an antibody mimetic, an aptamer, or anucleic acid molecule. In some embodiments of any of the providedmethods, the agent is selected from minocycline, naloxone, nimodipine,Riluzole, MOR103, lenalidomide, a cannabinoid (optionally WIN55 or212-2), intravenous immunoglobulin (IVIg), ibudilast, anti-miR-155locked nucleic acid (LNA), MCS110, PLX-3397, PLX647, PLX108-D1, PLX7486,JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945, emactuzumab, IMC-CS4, FPA008,LY-3022855, AMG-820 and TG-3003.

In particular embodiments of any of the provided methods, the agent isan inhibitor of colony stimulating factor 1 receptor (CSF1R). In certainembodiments of any of the provided methods, the inhibitor is selectedfrom: PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141,ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945 or a pharmaceutical salt or prodrugthereof; emactuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003or is an antigen-binding fragment thereof; or a combination of any ofthe foregoing.

In some embodiments of any of the provided methods, the inhibitor isPLX-3397. In particular embodiments of any of the provided methods, therecombinant receptor specifically binds to an antigen associated withthe disease or condition or expressed in cells of the environment of alesion associated with the disease or condition. In certain embodimentsof any of the provided methods, the disease or condition is a cancer. Insome embodiments of any of the provided methods, the disease orcondition is a myeloma, leukemia or lymphoma. In particular embodimentsof any of the provided methods, the disease or condition is a B cellmalignancy and/or is acute lymphoblastic leukemia (ALL), adult ALL,chronic lymphoblastic leukemia (CLL), non-Hodgkin lymphoma (NHL), andDiffuse Large B-Cell Lymphoma (DLBCL).

In certain embodiments of any of the provided methods, the antigen isROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX),tEGFR, Her2/neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20,CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folatereceptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelialglycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2,erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate binding protein(FBP), FCRLS, FCRHS, fetal acetylcholine receptor, GD2, GD3, HMW-MAA,IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor (kdr), kappalight chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM),Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentiallyexpressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AIMAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8,avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2Dligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin,murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100,oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA),Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), acyclin, cyclin A2, CCL-1, CD138, G Protein Coupled Receptor 5D (GPCRSD),or a pathogen-specific antigen.

In some embodiments of any of the provided methods, the recombinantreceptor is a T cell receptor or a functional non-T cell receptor. Inparticular embodiments of any of the provided methods, the recombinantreceptor is a chimeric antigen receptor (CAR). In certain embodiments ofany of the provided methods, the CAR comprises an extracellularantigen-recognition domain that specifically binds to the antigen and anintracellular signaling domain comprising an ITAM, wherein optionally,the intracellular signaling domain comprises an intracellular domain ofa CD3-zeta (CD3ζ) chain; and/or wherein the CAR further comprises acostimulatory signaling region, which optionally comprises a signalingdomain of CD28 or 4-1BB.

In some embodiments of any of the provided methods, the engineered cellscomprise T cells, optionally CD4⁺ and/or CD8⁺. In particular embodimentsof any of the provided methods, the T cells are primary T cells obtainedfrom a subject. In certain embodiments of any of the provided methods,the cell therapy comprises the administration of from or from about1×10⁵ to 1×10⁸ total recombinant receptor-expressing cells, total Tcells, or total peripheral blood mononuclear cells (PBMCs), from or fromabout 5×10⁵ to 1×10⁷ total recombinant receptor-expressing cells, totalT cells, or total peripheral blood mononuclear cells (PBMCs) or from orfrom about 1×10⁶ to 1×10⁷ total recombinant receptor-expressing cells,total T cells, or total peripheral blood mononuclear cells (PBMCs), eachinclusive.

In some embodiments of any of the provided methods, the cell therapycomprises the administration of no more than 1×10⁸ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 1×10⁷ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 0.5×10⁷ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 1×10⁶ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs), no more than 0.5×10⁶ total recombinantreceptor-expressing cells, total T cells, or total peripheral bloodmononuclear cells (PBMCs).

In particular embodiments of any of the provided methods, the dose thatis not associated with risk of developing toxicity or severe toxicity isor comprises less than or less than about 5×10⁷ total recombinantreceptor-expressing cells, optionally CAR⁺ cells, total T cells, ortotal peripheral blood mononuclear cells (PBMCs), such as less than orless than about 2.5×10⁷, less than or less than about 1.0×10⁷, less thanor less than about 5.0×10⁶, less than or less than about 1.0×10⁶, lessthan or less than about 5.0×10⁵, or less than or less than about 1×10⁵total recombinant receptor-expressing cells, optionally CAR⁺ cells,total T cells, or total peripheral blood mononuclear cells (PBMCs).

In certain embodiments of any of the provided methods, the dose that isnot associated with risk of developing toxicity or severe toxicity is orcomprises from or from about 1×10⁵ to 5×10⁷ total recombinantreceptor-expressing cells, optionally CAR⁺ cells, total T cells, ortotal peripheral blood mononuclear cells (PBMCs), such as 1×10⁵ to2.5×10⁷, 1×10⁵ to 1.0×10⁷, 1×10⁵ to 5.0×10⁶, 1×10⁵ to 1.0×10⁶, 1.0×10⁵to 5.0×10⁵, 5.0×10⁵ to 5×10⁷, 5×10⁵ to 2.5×10⁷ 5×10⁵ to 1.0×10⁷ 5×10⁵ to5.0×10⁶ 5×10⁵ to 1.0×10⁶ 1.0×10⁶ to 5×10⁷, 1×10⁶ to 2.5×10⁷, 1×10⁶ to1.0×10⁷, 1×10⁶ to 5.0×10⁶, 5.0×10⁶ to 5×10⁷, 5×10⁶ to 2.5×10⁷, 5×10⁶ to1.0×10⁷, 1.0×10⁷ to 5×10⁷, 1×10⁷ to 2.5×10⁷ or 2.5×10⁷ to 5×10⁷ totalrecombinant receptor-expressing cells, optionally CAR⁺ cells, total Tcells, or total peripheral blood mononuclear cells (PBMCs). In someembodiments of any of the provided methods, the engineered cells areautologous to the subject. In particular embodiments of any of theprovided methods, the engineered cells are allogeneic to the subject. Incertain embodiments of any of the provided methods, the reagent isdetectably labeled, optionally fluorescently labeled.

In some embodiments, the instructions provide information about athreshold level, individually for each of the one or more analytes, thatis indicative of whether a subject is likely to exhibit a response totreatment with the cell therapy. In some embodiments, the instructionsprovide information about a threshold level, individually for each ofthe one or more analytes, that is indicative of whether a subject islikely to exhibit a durable response following administration of thecell therapy. In some embodiments, the instructions provide informationabout a threshold level, individually for each of the one or moreanalytes, that is indicative of whether a subject is likely to exhibit atoxicity following administration of the cell therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the percentage of subjects who experienced laboratoryabnormalities and treatment-emergent adverse events (TEAEs) thatoccurred in ≥20% of subjects. *: One Grade 5 AE of multi-organ failureunrelated to study treatment and due to progression of lymphoma; †: OneGrade 5 AE of diffuse alveolar damage, investigator assessed as relatedto fludarabine, cyclophosphamide, and CAR T cell therapy, occurred onday 23 in a subject who refused mechanical ventilation for progressiverespiratory failure while neutropenic on growth factors and broadspectrum antibiotics and antifungals

FIG. 2 is a Kaplan meier curve depicting observed time to onset of CRSand neurotoxicity.

FIG. 3A and FIG. 3B depict 3 month objective response rates (ORR) amongsubgroups of treated subjects.

FIG. 4A and FIG. 4B show the duration of response (CR/PR, CR or PR) andoverall survival in the full and core cohort of subjects.

FIG. 5A shows the pharmacokinetics of the CAR⁺ T cells in peripheralblood at various time points post-treatment at different dose levels.FIG. 5B shows the pharmacokinetics of the CAR⁺ T cells in peripheralblood at various time points post-treatment between responders andnonresponders. FIG. 5C shows the pharmacokinetics of the CAR⁺ T cells inperipheral blood at various time points post-treatment in subjects thatdid or did not develop any neurotoxicity.

FIG. 6A shows the number of CD3⁺/CAR⁺, CD4⁺/CAR⁺, CD8⁺/CAR⁺ T cells inperipheral blood of a subject with chemorefractory transformed DLBCLmeasured at certain time points. FIG. 6B depicts a pretreatment axialPET-CT image showing an intracranial abnormality in the right middlecranial foss and extensive abnormality in subcutaneous tissues in theright posterior auricular region. FIG. 6C is a post-treatment PET-CTimage depicting resolution of the abnormality in FIG. 2B after treatmentwith anti-CD19 CAR⁺ T cells. FIG. 6D is a pretreatment brain MRI(high-resolution Ti-weighted image with the use of contrast material;axial view) showing a homogeneously enhancing mass in the right middlecranial fossa. FIG. 6E is a post-treatment MRI image showingnear-complete resolution of the enhancing mass. FIG. 6F is an axialPET-CT image at relapse showing right posteriour auricular tumorrecurrence associated with intense uptake of ¹⁸F-flurodeoxyglycose(arrow). FIG. 6G is a PET-CT imaging showing resolution of the posteriorauricular tumor after incisional biopsy and re-expansion of CAR⁺ Tcells.

FIG. 7 shows levels of analytes measured in the serum of subjects priorto administration of the CAR⁺ T cells and correlation to the developmentof neurotoxicity.

FIG. 8 shows a graph plotting progression-free time (months) andindicating best overall response and response durability, and individualclinical outcomes observed over time in individual subjects within aFull cohort and a Core cohort of NHL subjects treated with an anti-CD19cell therapy containing CAR-T-expressing CD4⁺ and CD8⁺ T cells. ^(a):Patients achieved BOR at month 1 except where otherwise noted; ^(b):Complete resolution of CNS involvement by lymphoma observed in 2patients; ^(c): One patient re-expanded after biopsy upon diseaseprogression

FIG. 9A depicts the median (±quartiles) number of CAR-expressing CD3⁺cells/μL, blood, assessed by flow cytometry using an antibody specificfor a truncated receptor (CD3, circle; N=87); or median (±quartiles)number of copies integrated CAR transgene/μg genomic DNA, assessed byquantitative polymerase chain reaction (qPCR) using primers specific fora woodchuck hepatitis virus post-transcriptional regulatory element(WPRE) present in the vector encoding the CAR (qPCR, square; N=85) inblood samples from 87 subjects that have been administered anti-CD19CAR-expressing cells. The cutoff for CAR⁺ cell detection in flowcytometry was set at ≥25 events in the CAR⁺ gate, and limit of detectionfor qPCR was ≥12.5 copies of CAR transgene perm of genomic DNA.

FIG. 9B depicts the relative numbers of CD4⁺ and CD8⁺ CAR-expressingcells/μL in blood and bone marrow samples from 67 subjects that havebeen administered anti-CD19 CAR-expressing cells, on day 11±3 days. Theline represents the line of unity and is not a regression line.

FIGS. 10A and 10B depict the median (±quartiles) area under the curvebetween days 0 and 28 (AUC₀₋₂₈; FIG. 10A) and maximum serumconcentration (C_(max); CAR⁺ cells/μL, blood; FIG. 10B) of CD4⁺ and CD8⁺CAR⁺ cells in subject subgroups with diffuse large B-cell lymphoma denovo or transformed from indolent lymphoma (DLBCL, NOS; N=27),transformed follicular lymphoma (tFL; N=10), DLBCL transformed frommarginal zone lymphoma or chronic lymphocytic leukemia (tMZL/tCLL; N=4),or mantle cell lymphoma (MCL; N-5), who have received CAR-expressing Tcells at DL1.

FIGS. 11A and 11B depict the median (±quartiles) area under the curvebetween days 0 and 28 (AUC₀₋₂₈; FIG. 11A) and maximum serumconcentration (C_(max); CAR⁺ cells/μL, blood; FIG. 11B) of CD3⁺, CD4⁺and CD8⁺ CAR⁺ cells in subjects who have received CAR⁺ cells at DL1 orDL2.

FIGS. 12A-12D depict the median (±quartiles) number of CAR-expressingCD4⁺ and CD8⁺ CAR⁺ cells/μL, blood over time, in subjects that developedcytokine release syndrome (any CRS) compared to subjects that have notdeveloped CRS (no CRS) (CD4⁺: FIG. 12A; CD8⁺: FIG. 12B) or in subjectsthat developed neurotoxicity (any NT) compared to subjects that have notdeveloped NT (no NT) (CD4⁺: FIG. 12C; CD8⁺: FIG. 12D).

FIGS. 13A and 13B depict the number of peak CD3⁺ CAR⁺ (CD3⁺ C_(max)) insubjects grouped by subjects who had the best overall response (BOR) ofCR, PR or PD, or a 3-month (M3) durable response of CR, PR or PD.

FIG. 14A depicts pre-lymphodepletionblood analyte levels in serumsamples from subjects that exhibited high CAR⁺ cell expansion (CD3⁺C_(max)>500) and subjects that exhibited low CAR⁺ cell expansion (CD3⁺C_(max)<500).

FIG. 14B depicts the peak blood analyte levels in serum samples fromsubjects that exhibited high CAR⁺ cell expansion (CD3⁺ C_(max)>500) andsubjects that exhibited low CAR⁺ cell expansion (CD3⁺ C_(max)<500).

FIG. 15 depicts a plot depicting pre-lymphodepletion SPD (cm²) againstAUC₀₋₂₈ (cells*day/μL) of CD3⁺ CAR⁺ cells, for individual subjectsadministered DL1 or DL2 of CAR⁺ cells.

FIGS. 16A and 16B depict pre-lymphodepletion blood analyte levels inserum samples from subjects that developed cytokine release syndrome(CRS grade 1-4) compared to subjects that have not developed CRS (CRSgrade 0) (FIG. 16A) or in subjects that developed neurotoxicity (NTgrade 0) compared to subjects that have not developed NT (NT grade 1-4)(FIG. 16B). The units were: Ferritin and D-dimer (μg/L); CRP (mg/L) andcytokines (pg/mL).

FIG. 17 depicts the assessment of pre-lymphodepletion patient parametersum of product dimensions (SPD; cm²), indicative of tumor burden, andlactate dehydrogenase (LDH; U/L) level, in subjects that developedcytokine release syndrome (any CRS) compared to subjects that have notdeveloped CRS (no CRS) or in subjects that developed neurotoxicity (anyNT) compared to subjects that have not developed NT (no NT).

FIG. 18A is a plot depicting pre-lymphodepletion SPD (cm²) againstpre-lymphodepletion LDH (U/L) levels, in individuals that have developedneurotoxicity (Grade 1-4 NT) or subjects that have not developed NT(Grade 0 NT) (left panel), and in individuals that have developed CRS(Grade 1-4 CRS) or subjects that have not developed CRS (Grade 0 CRS)(right panel). Dotted lines represent levels of SPD (50 cm² or higher)or LDH (500 U/L or higher) that is associated with higher rates of CRSor NT. FIG. 18B depicts the odds ratio estimates for developing CRS orNT based on the levels of SPD (50 cm² or higher) or LDH (500 U/L orhigher), with 95% confidence intervals (CI). FIG. 18C depicts the oddsratio estimates for developing CRS or NT based on the levels of SPD orLDH, including the odds ratio estimates for values lower than thethreshold, with 95% confidence intervals (CI).

FIG. 19 depicts pre-lymphodepletion tumor burden parameter (SPD) andblood analyte levels in for subjects that had a durable response at 3months versus for subjects that did not have a response at 3 months. Theunits were: Ferritin and D-dimer (μg/L); CRP and SAA-1 (mg/L) andcytokines (pg/mL).

FIGS. 20A and 20B depict peak blood analyte levels in serum samples fromsubjects that developed cytokine release syndrome (any CRS) compared tosubjects that have not developed CRS (no CRS) (FIG. 20A) or in subjectsthat developed neurotoxicity (any NT) compared to subjects that have notdeveloped NT (no NT) (FIG. 20B). The units were: CRP (mg/L), SAA-1(mg/L) and cytokines (pg/mL).

FIG. 21A depicts peak blood analyte levels in serum samples fromsubjects that had a best overall response (BOR) of complete response(CR) or partial response (PR) (N=57) compared to levels in subjects thathad stable disease (SD) or progressive disease (PD) (N=17). FIG. 21Bdepicts peak blood analyte levels in serum samples from subjects thathad a 3-month response of SD/PD (N=31), compared to subjects who had a3-month response CR/PR (N=35). The units were: CRP (mg/L), SAA-1 (mg/L)and cytokines (pg/mL).

FIG. 22 depicts month 3 objective response rates (ORR) among subgroupsof treated subjects, with the 95% confidence interval.

FIGS. 23A and 23B depict the duration of response (DOR) for the fullcohort (FIG. 23A) and the core cohort (FIG. 23B), and FIGS. 23C and 23Ddepict the overall survival for the full cohort (FIG. 23C) and the corecohort (FIG. 23D), for subjects who achieved CR, PR, all subjects thatshowed a response, non-responders, and all treated subjects. Median F/Uwas 6.3 months for duration of response.

FIG. 24 shows the percentage of subjects who experiencedtreatment-emergent adverse events (TEAEs) in the FULL DLBCL cohortoccurring in ≥20% of patients. Data for 5 patients with MCL treated withconforming product at DL1 with at least 28 days of follow-up were notincluded. ^(b): One grade 5 AE of septic shock unrelated to CAR⁺ T celladministration. ^(c): One grade 5 AE of diffuse alveolar damage,investigator assessed as related to fludarabine, cyclophosphamide, andCAR⁺ T cells, occurred on day 23 in a patient who refused mechanicalventilation for progressive respiratory failure while neutropenic ongrowth factors and broad-spectrum antibiotics and antifungals. ^(d):Laboratory anomalies.

FIG. 25 shows the percentage of subjects who developed CRS orneurotoxicity over time, in the full cohort.

FIG. 26 shows box plots displaying the T cell purity of T cellcompositions enriched for CD4⁺ and CD8⁺ cells at different stages of theprocess for generating engineered cell compositions containing CAR Tcells that is described in Example 8. The frequency (% of totalleukocytes) of CD4⁺ and CD8⁺ cells in the compositions are shown.

FIGS. 27A-27C show box plots displaying the concentration (FIG. 27A),viability (FIG. 27B), and frequency of caspase-3 negative (FIG. 27C)CD4⁺ and CD8⁺ CAR⁺ T cells in therapeutic cell compositions of a high orlow formulation volume.

FIG. 28A shows the number of CD3⁺ CAR⁺ T cells present in CAR T cellcompositions for administration at DL1 and DL2. FIG. 28B shows thenumber of CD4⁺ CAR⁺ and CD8⁺ CAR⁺ cells, and CD4⁺CAR⁺TNF-α+ cells andCD8⁺ CAR⁺TNF-α⁺ cells present in CAR T cell compositions foradministration at DL1 and DL2.

FIG. 29 shows the percentage of subjects who experiencedtreatment-emergent adverse events (TEAEs) in the FULL DLBCL cohortoccurring in ≥20% of the subject at a study time point described inExample 6. Data for 6 subjects with MCL treated with conforming productat DL1 with at least 28 days of follow-up were not included. ^(b): Onegrade 5 AE of septic shock unrelated to CAR⁺ T cell administration,occurred in the setting of disease progression. ^(c): One grade 5 AE ofdiffuse alveolar damage, investigator assessed as related tofludarabine, cyclophosphamide, and CAR⁺ T cells, occurred on day 23 in apatient who refused mechanical ventilation for progressive respiratoryfailure while neutropenic on growth factors and broad-spectrumantibiotics and antifungals. ^(d): Laboratory anomalies.

FIG. 30 depict the six (6) month objective response rates (ORR) amongsubgroups of treated subjects, with the 95% confidence interval. ^(a)Includes all DLBCL subjects treated at all dose levels in the COREcohort.

FIGS. 31A and 31B depict the duration of response (DOR) for the fullcohort (FIG. 31A) and the core cohort (FIG. 31B), and FIGS. 31C and 31Ddepict the overall survival for the full cohort (FIG. 31C) and the corecohort (FIG. 31D), for subjects who achieved CR, PR, all subjects thatshowed a response, non-responders, and all treated subjects. NE, notestimable.

DETAILED DESCRIPTION

I. Methods and Uses of Cell Therapy with Genetically Engineered Cells

Provided are methods and uses of engineered cells (e.g., T cells) and/orcompositions thereof, for the treatment of subjects having a disease orcondition, which generally is or includes a cancer or a tumor, such as aleukemia or a lymphoma, most particularly a non-Hodgkin lymphoma (NHL).In some aspects, the methods and uses provide for or achieve improvedresponse and/or more durable responses or efficacy and/or a reduced riskof toxicity or other side effects, e.g., in particular groups ofsubjects treated, as compared to certain alternative methods. In someembodiments, the methods are advantageous by virtue of theadministration of specified numbers or relative numbers of theengineered cells, the administration of defined ratios of particulartypes of the cells, treatment of particular patient populations, such asthose having a particular risk profile, staging, and/or prior treatmenthistory, and/or combinations thereof. Also provided are methods thatinclude assessing particular parameters, e.g., expression of specificbiomarkers or analytes, that can be correlated with development oftoxicity, and methods for treatment, e.g., intervention therapy, toprevent and/or ameliorate toxicities. Also provided are methods thatinvolve assessing particular parameters, e.g., expression of specificbiomarkers or analytes, that can be correlated with an outcome, such asa therapeutic outcome, including a response, such as a complete response(CR) or a partial response (PR), optionally durable response, such as aresponse that is durable for at least 3 months, 6 months or more; or asafety outcome, such as a development of a toxicity, for example,neurotoxicity or CRS, after administration of an immunotherapy and/orcell therapy. Also provided are methods to assess the likelihood ofresponse and/or likelihood of risk of toxicity, based on assessment ofthe parameters, such as expression of biomarkers or analytes. Alsoprovided are compositions for use in cell therapy. Also provided arearticles of manufacture and kits, e.g., for use in the methods providedherein. In some embodiments, the articles of manufacture and kitsoptionally contain instructions for using, according to the methodsprovided herein.

In some embodiments, the methods and uses include administering to thesubject cells expressing genetically engineered (recombinant) cellsurface receptors in adoptive cell therapy, which generally are chimericreceptors such as chimeric antigen receptors (CARs), recognizing anantigen expressed by, associated with and/or specific to the leukemia orlymphoma and/or cell type from which it is derived. The cells aregenerally administered in a composition formulated for administration;the methods generally involve administering one or more doses of thecells to the subject, which dose(s) may include a particular number orrelative number of cells or of the engineered cells, and/or a definedratio or compositions of two or more sub-types within the composition,such as CD4 vs. CD8 T cells.

In some embodiments, the cells, populations, and compositions areadministered to a subject having the particular disease or condition tobe treated, e.g., via adoptive cell therapy, such as adoptive T celltherapy. In some embodiments, the methods involve treating a subjecthaving a lymphoma or a leukemia, such as a non-Hodgkin lymphoma (NHL)with a dose of antigen receptor-expressing cells (e.g. CAR-expressingcells).

In some embodiments, the provided methods involve treating a specificgroup or subset of subjects, e.g., subjects identified as havinghigh-risk disease, e.g., high-risk NHL. In some aspects, the methodstreat subjects having a form of aggressive and/or poor prognosis B-cellnon-Hodgkin lymphoma (NHL), such as NHL that has relapsed or isrefractory (R/R) to standard therapy has a poor prognosis. In somecases, the overall response rate (ORR; also known in some cases asobjective response rate) to available therapies, to a standard of care,or to a reference therapy for the disease and/or patient population forwhich the therapy is indicated, is less than 40% and/or the completeresponse (CR; also known in some cases as complete remission) is lessthan 20%. In some embodiments, in chemorefractory DLBCL, the ORR with areference or available treatment or standard-of-care therapy is about26% and the CR rate is about 8% (Crump et al. Outomes in refractoryaggressive diffuse large B-cell lymphoma (DLBCL): Results from theinternational SCHOLAR study. ASCO 2016 [Abstract) 7516]). In someaspects, the provided methods, compositions, uses and articles ofmanufacture achieve improved and superior responses to availabletherapies.

In some embodiments, the methods, uses and articles of manufactureinvolve, or are used for treatment of subjects involving, selecting oridentifying a particular group or subset of subjects, e.g., based onspecific types of disease, diagnostic criteria, prior treatments and/orresponse to prior treatments. In some embodiments, the methods involvetreating a subject having relapsed following remission after treatmentwith, or become refractory to, one or more prior therapies; or a subjectthat has relapsed or is refractory (R/R) to one or more prior therapies,e.g., one or more lines of standard therapy. In some embodiments, themethods involve treating subjects having diffuse large B-cell lymphoma(DLBCL), not otherwise specified (NOS; de novo and transformed fromindolent), primary mediastinal B-cell lymphoma (PMBCL) or follicularlymphoma, such as follicular lymphoma grade 3B (FL3B). In someembodiments, the methods involve treating a subject that has an EasternCooperative Oncology Group Performance Status (ECOG) of 0-1 or 0-2. Insome embodiments, the methods treat a poor-prognosis population or ofDLBCL patients or subject thereof that generally responds poorly totherapies or particular reference therapies, such as one having one ormore, such as two or three, chromosomal translocations (such asso-called “double-hit” or “triple-hit” lymphoma; having translocationsMYC/8q24 loci, usually in combination with the t(14; 18) (q32; q21)bcl-2 gene or/and BCL6/3q27 chromosomal translocation; see, e.g., Xu etal. (2013) Int J Clin Exp Pathol. 6(4): 788-794), and/or one havingrelapsed, optionally relapsed within 12 months, following administrationof an autologous stem cell transplant (ASCT), and/or one having beendeemed chemorefractory.

In some aspects, the provided embodiments are based on observations thatthe provided methods can be used to achieve a high response rate withhigh durability, compared to certain available methods for cell therapy,without an increased risk of toxicity. In some embodiments, the providedmethods permit prolonged persistence of adoptively transferred cells forcell therapy, and/or low rate of developing toxicity in the subject. Insome embodiments, the methods can be used to select subjects fortreatment with cell therapy that are likely or more likely to respond tothe therapy and/or to determine appropriate doses or dosing regime forhigher response rate and/or more durable response, while minimizing therisk of toxicity. Such methods can inform rational strategies tofacilitate the safe and effective clinical application of adoptive celltherapy, such as CAR-T cell therapy.

In some embodiments, the antigen receptor (e.g. CAR) specifically bindsto a target antigen associated with the disease or condition, such asassociated with NHL. In some embodiments, the antigen associated withthe disease or disorder is selected from CD20, CD19, CD22, ROR1, CD45,CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments, the methods include administration of the cells ora composition containing the cells to a subject, tissue, or cell, suchas one having, at risk for, or suspected of having the disease,condition or disorder. In some embodiments, the subject is the subjectis an adult. In some embodiments, the subject is over at or about 30,40, 50, 60, or 70 years of age.

In some embodiments, the methods include administration of cells to asubject selected or identified as having a certain prognosis or risk ofNHL. Non-Hodgkin lymphoma (NHL) is a can be a variable disease. Somesubjects with NHL may survive without treatment while others may requireimmediate intervention. In some cases, subjects with NHL may beclassified into groups that may inform disease prognosis and/orrecommended treatment strategy. In some cases, these groups may be “lowrisk,” “intermediate risk,” “high risk,” and/or “very high risk” andpatients may be classified as such depending on a number of factorsincluding, but not limited to, genetic abnormalities and/ormorphological or physical characteristics. In some embodiments, subjectstreated in accord with the methods, and/or with the articles ofmanufacture or compositions, are classified or identified based on therisk of NHL. In some embodiments, the subject is one that has high riskNHL.

In some embodiments, the subject has been previously treated with atherapy or a therapeutic agent targeting the disease or condition, e.g.,NHL, prior to administration of the cells expressing the recombinantreceptor. In some embodiments, the subject has been previously treatedwith a hematopoietic stem cell transplantation (HSCT), e.g., allogeneicHSCT or autogeneic HSCT. In some embodiments, the subject has had poorprognosis after treatment with standard therapy and/or has failed one ormore lines of previous therapy. In some embodiments, the subject hasbeen treated or has previously received at least or about at least orabout 1, 2, 3, or 4 other therapies for treating the NHL other than alymphodepleting therapy and/or the dose of cells expressing the antigenreceptor. In some embodiments, the subject has been previously treatedwith chemotherapy or radiation therapy. In some aspects, the subject isrefractory or non-responsive to the other therapy or therapeutic agent.In some embodiments, the subject has persistent or relapsed disease,e.g., following treatment with another therapy or therapeuticintervention, including chemotherapy or radiation.

In some embodiments, the subject is one that is eligible for atransplant, such as is eligible for a hematopoietic stem celltransplantation (HSCT), e.g., allogeneic HSCT. In some such embodiments,the subject has not previously received a transplant, despite beingeligible, prior to administration of the engineered cells (e.g. CAR-Tcells) or a composition containing the cells to the subject as providedherein.

In some embodiments, the subject is one that is not eligible for atransplant, such as is not eligible for a hematopoietic stem celltransplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, sucha subject is administered the engineered cells (e.g. CAR-T cells) or acomposition containing the cells according to the provided embodimentsherein.

In some embodiments, the methods include administration of cells to asubject selected or identified as having high-risk NHL. In someembodiments, the subject exhibits one or more cytogenetic abnormalities,such as associated with high-risk NHL. In some embodiments, the subjectis selected or identified based on having a disease or conditioncharacterized or determined to be aggressive NHL, diffuse large B celllymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), Tcell/histocyte-rich large B cell lymphoma (TCHRBCL), Burkitt's lymphoma,mantle cell lymphoma (MCL), and/or follicular lymphoma (FL). Inparticular embodiments, the subject to be treated using the methodsprovided herein include subjects with aggressive NHL, in particular,with diffuse large B-cell lymphoma (DLBCL), not otherwise specified(NOS; de novo and transformed from indolent), primary mediastinal B-celllymphoma (PMBCL) or follicular lymphoma grade 3B (FL3B). In someembodiments, the subject has poor performance status. In some aspects,the population to be treated includes subjects having an EasternCooperative Oncology Group Performance Status (ECOG) that is anywherefrom 0-2. In other aspects of any of the embodiments, the subjects to betreated included ECOG 0-1 or do not include ECOG 2 subjects. In someaspects of any of the embodiments, the subjects to be treated havefailed two or more prior therapies. In some embodiments, the subjectdoes not have DLBCL transformed from marginal zone lymphoma (MZL) andchronic lymphocytic leukemia (CLL; Richter's). In some embodiments, asubject with CLL can exhibit Richter's syndrome (RS), defined as thetransformation of CLL into an aggressive lymphoma, most commonly diffuselarge B-cell lymphoma (DLBCL) (see, e.g., Parikh et al. Blood 2014123:1647-1657). In some embodiments, the subject has mantle celllymphoma (MCL). In some embodiments, the subject has features thatcorrelate with poor overall survival. In some embodiments, the subjecthas never achieved a complete response (CR), never received autologousstem cell transplant (ASCT), refractory to 1 or more second linetherapy, has primary refractory disease, and/or has an ECOG performancescore of 2.

In some embodiments, the subject to be treated includes a group ofsubjects with diffuse large B-cell lymphoma (DLBCL), de novo ortransformed from indolent lymphoma (NOS), primary mediastinal largeb-cell lymphoma (PMBCL), and follicular lymphoma grade 3b (FL3B) afterfailure of 2 lines of therapy, and ECOG score of 0-2, and the subjectmay optionally have previously been treated with allogeneic stem celltransplantation (SCT). In some embodiments, such subject group can bereferred to as the “full cohort.” In some embodiments, the subject isselected for treatment with adoptive cell therapy, if the subject meetssaid criteria. In some embodiments, within said group (“full cohort”),the subject is not selected for treatment or excluded from treatment, ifthe subject has a poor performance status (e.g. ECOG 2) and/or has DLBCLtransformed from marginal zone lymphomas (MZL) and chronic lymphocyticleukemia (CLL, Richter's). Thus, in some embodiments, the subject isselected for treatment if the subject has subjects with diffuse largeB-cell lymphoma (DLBCL), de novo or transformed from indolent lymphoma(NOS), primary mediastinal large b-cell lymphoma (PMBCL), and follicularlymphoma grade 3b (FL3B) after failure of 2 lines of therapy, and ECOGscore of 0 or 1, and the subject may optionally have previously beentreated with allogeneic stem cell transplantation (SCT) but does nothave DLBCL transformed from marginal zone lymphomas (MZL) and chroniclymphocytic leukemia (CLL, Richter's). In some embodiments, such subjectgroup can be referred to as the “core cohort.” In some embodiments, thesubject to be treated is subjects in the “core cohort.”

In some aspects, the provided embodiments are based on observations thatcertain subject population, for example, the “core cohort” subjects whohave been administered a certain dose of the cell therapy, show anoverall response rate (ORR) of more than 80%, with a complete response(CR) rate of more than 55%, with high durability, e.g., response that ismaintained over a longer period of time, e.g., more than 3 months, witha 3-month ORR of over 65%, and a 3-month CR rate of approximately 50%.In particular, the provided observations indicated that the 3-month ORRwas high in subjects with two or three chromosomal translocations(“double-hit” or “triple-hit” lymphoma; having translocations MYC/8q24loci, usually in combination with the t(14; 18) (q32; q21) bcl-2 geneor/and BCL6/3q27 chromosomal translocation; see, e.g., Xu et al. (2013)Int J Clin Exp Pathol. 6(4): 788-794), primary-refractory lymphomas,chemorefractory DLBCL, and subjects who have never previously achievedCR.

In some aspects, provided are compositions, methods and uses foradministration of a defined composition of the cell therapy, atparticular doses, that are associated with a high response rate and/orhigh durability of response, and low levels and/or incidence oftoxicity. In some embodiments, the composition or dose administered is aflat and/or fixed dose, such as a precise flat dose, of cells and/or ofone or more cells having a particular phenotype, such as a particularnumber of such cells or a number that is within a particular rangeand/or degree of variability or variance as compared to a target number.In some embodiments, the composition or dose administered contains adefined ratio of CD4⁺ and CD8⁺ cells (e.g., 1:1 ratio of CD4⁺:CD8⁺ CAR⁺T cells) and/or contains a ratio that is within a certain degree ofvariability from such ratio, such as no more than ±10%, such as no morethan ±8%, such as a degree of variability or variance of no more than±10%, such as no more than ±8%. In some embodiments, the CD4⁺ and CD8⁺cells are individually formulated and administered. In some embodiments,the administered cells exhibit consistent activity and/or function,e.g., cytokine production, apoptosis and/or expansion. In someembodiments, the provided compositions exhibit highly consistent anddefined activity, and low variability between cells, e.g., in terms ofcell number, cell function and/or cell activity, in the composition orbetween preparations. In some embodiments, the consistency in activityand/or function, e.g., low variability between preparations ofcompositions, allows improved efficacy and/or safety. In someembodiments, administration of the defined compositions resulted in lowproduct variability and low toxicity, e.g., CRS or neurotoxicity,compared to administration of cell compositions with high heterogeneity.In some embodiments, the defined, consistent composition also exhibitsconsistent cell expansion. Such consistency can facilitate theidentification of dose, therapeutic window, evaluation of dose reponseand identification of factors of the subject that may correlate withsafety or toxicity outcomes.

In some embodiments, in a certain cohort of subjects receiving a singleinfusion of a particular dose level, a durable response rate after 6months of greater than 60% can be achieved. In some embodiments, thesubjects in some cohorts can achieve an overall response rate (ORR, insome cases also known as objective response rate) of more than 80%, acomplete response (CR) rate of more than 60% and/or a high durable CRrate at 6 months. In some embodiments, subjects receiving a defined doseshow improved safety outcomes, e.g., more than two-thirds of thesubjects that do not exhibit any CRS or NT. In some aspects, the rate ofsevere CRS or severe NT is low. In some embodiments, a higher exposure(e.g., C_(max) and AUC₀₋₂₈) observed with a particular defined dose,does not associate with increased toxicity, e.g., CRS or NT. In someembodiments, particular factors of the subject, e.g., certainbiomarkers, can be used to predict the risk of toxicity. In someembodiments, the provided embodiments can be used to achieve highresponse rate with low risk of toxicity.

In some embodiments, no more than 25%, no more than 20%, no more than15%, no more than 10% or no more than 5% of subjects treated using theprovided compositions, articles of manufacture, kits, methods and usesare administered an agent (e.g. tocilizumab and/or dexamethasone) toameliorate, treat or prevent a toxicity, either prior to or subsequentto administration of the cell therapy. In some embodiments, the subjectis not administered any prophylaxis treatment prior to receiving theengineered cells (e.g. CAR-T cells).

In some embodiments, the provided embodiments provide an advantage,e.g., permits administration of the cell therapy on an outpatient basis.In some embodiments, the administration of the cell therapy, e.g. doseof T cells in accord with the provided embodiments, can be performed onan outpatient basis or does not require admission to the subject to thehospital, such as admission to the hospital requiring an overnight stay.In some embodiments, such outpatient administration can allow increasedaccess and decreased costs, while maintaining a high, durable responserate with low toxicity. In some aspects, outpatient treatment can beadvantageous for patients who already are otherwise immunocompromised byprior treatments, e.g. post-lympodepletion, and are at a greater riskfor exposures at a hospital stay or in an in-patient setting. In someaspects, outpatient treatments also increases options for treatment forsubjects who may not have access to in-patient, hospital settings, ortransplant centers, thereby expanding access to the treatment. In someembodiments, subjects treated on an outpatient basis using the providedcompositions, articles of manufacture, kits, methods and uses remain inoutpatient for at least 3 days or a certain percentage of subjects, e.g.at least 60%, at least 70%, at least 80%, at least 85%, at least 90% orat least 95%, of subjects so treated remain in outpatient for at least 3days. In some aspects, the subjects remain in outpatient for at least 4days, 5 days, 6 days, 7 days, 8 days or more. In some embodiments,subjects treated using the provided compositions, articles ofmanufacture, kits, methods and uses show a reduction in the duration ofhospital stay, e.g., of at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35% or at least 40%, compared tosubjects treated with other compositions, articles of manufacture, kits,methods and uses.

In some embodiments, the methods, cells and compositions can providehigh rate of durable response to subjects across a range of patientcharacteristics and/or tumor burden. In some embodiments, the methods,cells and compositions can provide high rate of durable response to highrisk patients with poor prognosis, with a reduced risk of adverseeffects or toxicities. In some embodiments, the methods and uses providefor or achieve a higher response rate and/or more durable responses orefficacy and/or a reduced risk of toxicity or other side effects thatcan be associated with cell therapy, such as neurotoxicity (NT) orcytokine release syndrome (CRS). In some aspects, the providedobservations indicated a low rate of severe NT (sNT) or severe CRS(sCRS), and a high rate of patients without any toxicities, e.g., NT orCRS.

In some embodiments, at least 35%, at least 40%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, or at least 75% or moreof the subjects treated according to the provided methods, and/or withthe provided articles of manufacture or compositions, achieve a completeresponse (CR). In some embodiments, at least 50%, at least 60%, at least70%, at least 80%, or at least 90% of the subjects treated according tothe provided methods, and/or with the provided articles of manufactureor compositions, achieve an objective response (OR). In someembodiments, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95% or more of the subjects treated according to the providedmethods, and/or with the provided articles of manufacture orcompositions, achieve a CR or OR by one month, by two months or by threemonths.

In some embodiments, by three months, four months, five months, sixmonths or more after initiation of administration of the cell therapy,at least 60%, at least 70%, at least 80%, at least 90%, at least 95% ormore of the subjects treated according to the provided methods, and/orwith the provided articles of manufacture or compositions, remain inresponse, such as remain in CR or OR. In some embodiments, suchresponse, such as CR or OR, is durable for at least three months, fourmonths, five months, six months, seven months, eight months or ninemonths, such as in at least or about at least 60%, at least 70%, atleast 80%, at least 90%, at least 95% or more of the subjects treatedaccording to the provided methods or in such subjects who achieve a CRby one month or by three months. In some embodiments, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95% or more of thesubjects treated according to the provided methods, and/or with theprovided articles of manufacture or compositions, or such subjects whoachieve a CR by one month or by three months, survive or survive withoutprogression for greater than or greater than about three months, fourmonths, five months, six months, seven months, eight months or ninemonths.

In some embodiments, the resulting response observed in such subjects bythe treatment in accord with the provided methods, and/or with theprovided articles of manufacture or compositions, is associated with orresults in a low risk of any toxicity or a low risk of severe toxicityin a majority of the subjects treated. In some embodiments, greater thanor greater than about 30%, 35%, 40%, 50%, 55%, 60% or more of thesubjects treated according to the provided methods and/or with theprovided articles of manufacture or compositions do not exhibit anygrade of CRS or any grade of neurotoxicity (NT). In some embodiments,greater than or greater than about 50%, 60%, 70%, 80% or more of thesubjects treated according to the provided methods and/or with theprovided articles of manufacture or compositions do not exhibit severeCRS or grade 3 or higher CRS. In some embodiments, greater than orgreater than about 50%, 60%, 70%, 80% or more of the subjects treatedaccording to the provided methods, and/or with the provided articles ofmanufacture or compositions, do not exhibit severe neurotoxicity orgrade 3 or higher neurotoxicity, such as grade 4 or 5 neurotoxicity.

In some embodiments, at least at or about 45%, 50%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of subjects treated according to the method and/orwith the provided articles of manufacture or compositions do not exhibitearly onset CRS or neurotoxicity and/or do not exhibit onset of CRSearlier than 1 day, 2 days, 3 days or 4 days following initiation of theadministration. In some embodiments, at least at or about 45%, 50%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% of subjects treated according tothe methods, and/or with the provided articles of manufacture orcompositions, do not exhibit onset of neurotoxicity earlier than 3 days,4 days, 5 days, six days or 7 days following initiation of theadministration. In some aspects, the median onset of neurotoxicity amongsubjects treated according to the methods, and/or with the providedarticles of manufacture or compositions, is at or after the median peakof, or median time to resolution of, CRS in subjects treated accordingto the method. In some cases, the median onset of neurotoxicity amongsubjects treated according to the method is greater than at or about 8,9, 10, or 11 days.

In some embodiments, such results are observed following administrationof from or from about 5×10⁷ to 1.5×10⁸, such as 5×10⁷ to 1×10⁸ totalrecombinant receptor-expressing T cells, such as a dose of T cellsincluding CD4⁺ and CD8⁺ T cells administered at a defined ratio asdescribed herein, e.g. at or about a 1:1 ratio, and/or at a precise orflat or fixed number of CAR⁺ T cells, or precise or flat or fixed numberof a particular type of CAR⁺ T cells such as CD4⁺CAR⁺ T cells and/orCD8⁺ CAR⁺ T cells, and/or a number of any of such cells that is within aspecified degree of variance, such as no more than, + or − (plus orminus, in some cases indicated as ±), 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,or 15% as compared to such precise or flat or fixed number. In someembodiments, such flat or fixed number of cells is at or about 2.5×10⁷,5×10⁷, 10×10⁷, 15×10⁷ or 20×10⁷, e.g., of total CAR⁺ T cells or of CD8⁺and/or CD4⁺ CAR⁺ T cells. In some embodiments, the number of cells inthe dose includes or consists of or consists essentially of 5×10⁷CD4⁺CAR⁺ T cells (optionally 2.5×10⁷ CD4⁺CAR⁺ T cells and 2.5×10⁷ CD8⁺CAR⁺ T cells); in some embodiments, it includes or consists of orconsists essentially of 10×10⁷ CAR⁺ T cells (optionally 5×10⁷ CD4⁺CAR⁺ Tcells and 5×10⁷ CD8⁺ CAR⁺ T cells). In some aspects, the number of cellsadministered, is within a certain degree of variance of such numbers inthe aforementioned embodiments, such as within plus or minus (±) 5, 6,7, 8, 9, or 10%, such as within plus or minus 8%, as compared to suchnumber(s) of cells. In some aspects, the dose is within a range in whicha correlation is observed (optionally a linear relationship) between thenumber of such cells (e.g., of total CAR⁺ T cells or of CD8⁺ and/or CD4⁺CAR⁺ T cells) and one or more outcomes indicative of therapeuticresponse, or duration thereof (e.g., likelihood of achieving aremission, a complete remission, and/or a particular duration ofremission) and/or duration of any of the foregoing. In some aspects, itis found that the higher dose of cells administered can result ingreater response without or without substantially impacting or affectingthe incidence or risk of toxicity (e.g. CRS or neurotoxicity), or degreeof incidence or risk of toxicity, in the subject e.g. severe CRS orsevere neurotoxicity.

In some aspects, the provided methods can achieve a high or a particularrate of response (such as a rate of response among a population asassessed after a certain period post-administration, such as threemonths or six months), e.g., ORR (such as a 6-month or 3-month ORR) of40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% ormore, 70% or more, 75% or 80% or 81%, 82%, 83%, 84% or 85% or more andCR rate (such as a 6-month or 3-month CR rate) of 30% or more, 35% ormore, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more,65% or more, 70% or more, 71%, 72%, 73% or more or approximately 75% ormore, which also is durable such as for a particular period of time orat least a particular period of time, e.g., is sustained for more than1, 3 or 6 months or more or 9 months or more after initiation oftherapy. In some embodiments, such rates of response and durability arereceived following only a single administration or dose of such therapy.Treatment of such subjects by the provided methods, and/or with theprovided articles of manufacture or compositions, in some embodiments,also result in the subjects achieving the high rate of response, yet notexhibiting higher incidence of developing toxicities, such asneurotoxicity or CRS, even at a higher cell dosage. In some embodiments,about or greater than 50%, 55% or 60% of subjects achieving suchresponses do not develop any grade of toxicity, such as any grade of CRSand/or neurotoxicity.

Thus, in some embodiments, the provided methods, articles of manufactureand/or compositions, can offer advantages over other available methodsor solutions or approaches for treatment such as for adoptive celltherapy. In particular, among the provided embodiments are those thatoffer an advantage for subjects with high-risk NHL, by achieving adurable response at a high rate, with reduced incidence of toxicities orside effects.

A. Method of Treatment

Provided herein are methods of treatment that involve administeringengineered cells or compositions containing engineered cells, such asengineered T cells. Also provided are methods and uses of engineeredcells (e.g., T cells) and/or compositions thereof, including methods forthe treatment of subjects having a disease or condition such as aleukemia or a lymphoma, e.g., a non-Hodgkin lymphoma (NHL), thatinvolves administration of the engineered cells and/or compositionsthereof. In some embodiments, the provided methods and uses can achieveimproved response and/or more durable responses or efficacy and/or areduced risk of toxicity or other side effects, e.g., in particulargroups of subjects treated, as compared to certain alternative methods.In some aspects, also provided are methods of administering engineeredcells or compositions containing engineered cells, such as engineered Tcells, to a subject, such as a subject that has a disease or disorder.In some aspects, also provided are uses of engineered cells orcompositions containing engineered cells, such as engineered T cells fortreatment of a disease or disorder. In some aspects, also provided areuses of engineered cells or compositions containing engineered cells,such as engineered T cells for the manufacture of a medicament for thetreatment of a disease or disorder. In some aspects, also provided aremethods of administering engineered cells or compositions containingengineered cells, such as engineered T cells, for use in treatment of adisease or disorder, or for administration to a subject having a diseaseor disorder. In some aspects, the uses of the engineered cells orcompositions containing engineered cells, such as engineered T cells arein accord with any of the methods described herein.

General methods for administration of cells for adoptive cell therapyare known and may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

The disease or condition that is treated can be any in which expressionof an antigen is associated with and/or involved in the etiology of adisease condition or disorder, e.g. causes, exacerbates or otherwise isinvolved in such disease, condition, or disorder. Exemplary diseases andconditions can include diseases or conditions associated with malignancyor transformation of cells (e.g. cancer), autoimmune or inflammatorydisease, or an infectious disease, e.g. caused by a bacterial, viral orother pathogen. Exemplary antigens, which include antigens associatedwith various diseases and conditions that can be treated, are describedabove. In particular embodiments, the chimeric antigen receptor ortransgenic TCR specifically binds to an antigen associated with thedisease or condition.

Among the diseases, conditions, and disorders are tumors, includingsolid tumors, hematologic malignancies, and melanomas, and includinglocalized and metastatic tumors, infectious diseases, such as infectionwith a virus or other pathogen, e.g., HIV, HCV, HBV, CMV, HPV, andparasitic disease, and autoimmune and inflammatory diseases. In someembodiments, the disease, disorder or condition is a tumor, cancer,malignancy, neoplasm, or other proliferative disease or disorder. Suchdiseases include but are not limited to leukemia, lymphoma, e.g., acutemyeloid (or myelogenous) leukemia (AML), chronic myeloid (ormyelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic)leukemia (ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia(HCL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL),Marginal zone lymphoma, Burkitt lymphoma, Hodgkin lymphoma (HL),non-Hodgkin lymphoma (NHL), Anaplastic large cell lymphoma (ALCL),follicular lymphoma, refractory follicular lymphoma, diffuse largeB-cell lymphoma (DLBCL) and multiple myeloma (MM). In some embodiments,disease or condition is a B cell malignancy selected from among acutelymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia(CLL), non-Hodgkin lymphoma (NHL), and Diffuse Large B-Cell Lymphoma(DLBCL). In some embodiments, the disease or condition is NHL and theNHL is selected from the group consisting of aggressive NHL, diffuselarge B cell lymphoma (DLBCL), NOS (de novo and transformed fromindolent), primary mediastinal large B cell lymphoma (PMBCL), Tcell/histocyte-rich large B cell lymphoma (TCHRBCL), Burkitt's lymphoma,mantle cell lymphoma (MCL), and/or follicular lymphoma (FL), optionallyfollicular lymphoma Grade 3B (FL3B).

In some embodiments, NHL can be staged based on the Luganoclassification (see, e.g., Cheson et al., (2014) JCO 32(27):3059-3067;Cheson, B. D. (2015) Chin Clin Oncol 4(1):5). In some cases, the stagesare described by Roman numerals I through IV (1-4), and limited stage (Ior II) lymphomas that affect an organ outside the lymph system (anextranodal organ) are indicated by an E. Stage I represents involvementin one node or a group of adjacent nodes, or a single extranodal lesionswithout nodal involvement (IE). Stage 2 represents involvement in two ormore nodal groups on the same side of the diaphragm or stage I or II bynodal extent with limited contiguous extranodal involvement (IIE). StageIII represents involvement in nodes on both sides of the diaphragm ornodes above the diaphragm with spleen involvement. Stage IV representsinvolvement in additional non-contiguous extralymphatic involvement. Inaddition, “bulky disease” can be used to describe large tumors in thechest, in particular for stage II. The extent of disease is determinedby positron emission tomography (PET)-computed tomography (CT) for avidlymphomas, and CT for non-avid histologies.

In some embodiments, the Eastern Cooperative Oncology Group (ECOG)performance status indicator can be used to assess or select subjectsfor treatment, e.g., subjects who have had poor performance from priortherapies (see, e.g., Oken et al. (1982) Am J Clin Oncol. 5:649-655).The ECOG Scale of Performance Status describes a patient's level offunctioning in terms of their ability to care for themselves, dailyactivity, and physical ability (e.g., walking, working, etc.). In someembodiments, an ECOG performance status of 0 indicates that a subjectcan perform normal activity. In some aspects, subjects with an ECOGperformance status of 1 exhibit some restriction in physical activitybut the subject is fully ambulatory. In some aspects, patients with anECOG performance status of 2 is more than 50% ambulatory. In some cases,the subject with an ECOG performance status of 2 may also be capable ofselfcare; see e.g., Sorensen et al., (1993) Br J Cancer 67(4) 773-775.The criteria reflective of the ECOG performance status are described inTable 1 below:

TABLE 1 ECOG Performance Status Criteria Grade ECOG performance status 0Fully active, able to carry on all pre-disease performance withoutrestriction 1 Restricted in physically strenuous activity but ambulatoryand able to carry out work of a light or sedentary nature, e.g., lighthouse work, office work 2 Ambulatory and capable of all selfcare butunable to carry out any work activities; up and about more than 50% ofwaking hours 3 Capable of only limited selfcare; confined to bed orchair more than 50% of waking hours 4 Completely disabled; cannot carryon any selfcare; totally confined to bed or chair 5 Dead

In some embodiments, the subject has or has been identified as having ashaving a double/triple hit lymphoma or a lymphoma of the double/triplehit molecular subtypes. In some embodiments, the lymphoma is a doublehit lymphoma characterized by the presence of MYC (myelocytomatosisoncogene), BCL2 (B-cell lymphoma 2), and/or BCL6 (B-cell lymphoma 6)gene rearrangements (e.g., translocations). In some embodiments, thegene rearrangement affects the MYC/8q24 locus in combination withanother gene rearrangement. For example, the other gene rearrangementincludes t(14;18)(q32;q21) involving BCL2. In some embodiments, the generearrangements affect the MYC/8q24 locus in combination with BCL6/3q27.In some embodiments, the lymphoma is a triple hit lymphoma characterizedby the presence of MYC, BCL2, and BCL6 gene rearrangements; see, e.g.,Aukema et al., (2011) Blood 117:2319-2331. In some aspects of suchembodiments the subject is ECOG 0-1 or does not have or is not suspectedor characterized as having DLBCL transformed from MZL or CLL. Inaspects, the therapy is indicated for such subjects and/or theinstructions indicate administration to a subject within suchpopulation. In some embodiments, based on the 2016 WHO criteria(Swerdlow et al., (2016) Blood 127(20):2375-2390), double/triple hitlymphoma can be considered high-grade B-cell lymphoma, with MYC and BCL2and/or BCL6 rearrangements with DLBCL histology (double/triple hit).

In some embodiments, the disease or condition is an infectious diseaseor condition, such as, but not limited to, viral, retroviral, bacterial,and protozoal infections, immunodeficiency, Cytomegalovirus (CMV),Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In someembodiments, the disease or condition is an autoimmune or inflammatorydisease or condition, such as arthritis, e.g., rheumatoid arthritis(RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatorybowel disease, psoriasis, scleroderma, autoimmune thyroid disease,Grave's disease, Crohn's disease, multiple sclerosis, asthma, and/or adisease or condition associated with transplant.

In some embodiments, the antigen associated with the disease or disorderis selected from among αvβ6 integrin (avb6 integrin), B cell maturationantigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known asCAIX or G250), a cancer-testis antigen, cancer/testis antigen 1B (CTAG,also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), acyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20,CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123,CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4),epidermal growth factor protein (EGFR), type III epidermal growth factorreceptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2),epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2(EPHa2), estrogen receptor, Fc receptor like 5 (FCRLS; also known as Fcreceptor homolog 5 or FCRHS), fetal acetylcholine receptor (fetal AchR),a folate binding protein (FBP), folate receptor alpha, ganglioside GD2,O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100),glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPCRSD), Her2/neu(receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbBdimers, Human high molecular weight-melanoma-associated antigen(HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen A1(HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domainreceptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM),CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A(LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3,MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus(CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D)ligands, melan A (MART-1), neural cell adhesion molecule (NCAM),oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME),progesterone receptor, a prostate specific antigen, prostate stem cellantigen (PSCA), prostate specific membrane antigen (PSMA), ReceptorTyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblastglycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72(TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 orgp75), Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factorreceptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific orpathogen-expressed antigen, or an antigen associated with a universaltag, and/or biotinylated molecules, and/or molecules expressed by HIV,HCV, HBV or other pathogens. Antigens targeted by the receptors in someembodiments include antigens associated with a B cell malignancy, suchas any of a number of known B cell marker. In some embodiments, theantigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33,Igkappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments, the antigen is or includes a pathogen-specific orpathogen-expressed antigen. In some embodiments, the antigen is a viralantigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterialantigens, and/or parasitic antigens.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by autologous transfer, in which the cells are isolatedand/or otherwise prepared from the subject who is to receive the celltherapy, or from a sample derived from such a subject. Thus, in someaspects, the cells are derived from a subject, e.g., patient, in need ofa treatment and the cells, following isolation and processing areadministered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by allogeneic transfer, in which the cells are isolatedand/or otherwise prepared from a subject other than a subject who is toreceive or who ultimately receives the cell therapy, e.g., a firstsubject. In such embodiments, the cells then are administered to adifferent subject, e.g., a second subject, of the same species. In someembodiments, the first and second subjects are genetically identical. Insome embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class orsupertype as the first subject.

The cells can be administered by any suitable means, for example, bybolus infusion, by injection, e.g., intravenous or subcutaneousinjections, intraocular injection, periocular injection, subretinalinjection, intravitreal injection, trans-septal injection, subscleralinjection, intrachoroidal injection, intracameral injection,subconjectval injection, subconjuntival injection, sub-Tenon'sinjection, retrobulbar injection, peribulbar injection, or posteriorjuxtascleral delivery. In some embodiments, they are administered byparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In some embodiments, a given dose isadministered by a single bolus administration of the cells. In someembodiments, it is administered by multiple bolus administrations of thecells, for example, over a period of no more than 3 days, or bycontinuous infusion administration of the cells. In some embodiments,administration of the cell dose or any additional therapies, e.g., thelymphodepleting therapy, intervention therapy and/or combinationtherapy, is carried out via outpatient delivery.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of cells orrecombinant receptors, the severity and course of the disease, whetherthe cells are administered for preventive or therapeutic purposes,previous therapy, the subject's clinical history and response to thecells, and the discretion of the attending physician. The compositionsand cells are in some embodiments suitably administered to the subjectat one time or over a series of treatments.

In some embodiments, the cells are administered as part of a combinationtreatment, such as simultaneously with or sequentially with, in anyorder, another or additional therapeutic intervention, such as anantibody or engineered cell or receptor or agent, such as a cytotoxic ortherapeutic agent. The cells in some embodiments are co-administeredwith one or more additional therapeutic agents or in connection withanother therapeutic intervention, either simultaneously or sequentiallyin any order. In some embodiments, the additional therapeutic agent isany interventions or agents described herein, such as any interventionsor agents descried that can ameliorate symptoms of toxicity describedherein, for example, in Section II. In some contexts, the cells areco-administered with another therapy sufficiently close in time suchthat the cell populations enhance the effect of one or more additionaltherapeutic agents, or vice versa. In some embodiments, the cells areadministered prior to the one or more additional therapeutic agents. Insome embodiments, the cells are administered after the one or moreadditional therapeutic agents. In some embodiments, the one or moreadditional agents include a cytokine, such as IL-2, for example, toenhance persistence. In some embodiments, the methods compriseadministration of a chemotherapeutic agent.

In some embodiments, the methods comprise administration of achemotherapeutic agent, e.g., a conditioning chemotherapeutic agent, forexample, to reduce tumor burden prior to the administration.

Preconditioning subjects with immunodepleting (e.g., lymphodepleting)therapies in some aspects can improve the effects of adoptive celltherapy (ACT).

Thus, in some embodiments, the methods include administering apreconditioning agent, such as a lymphodepleting or chemotherapeuticagent, such as cyclophosphamide, fludarabine, or combinations thereof,to a subject prior to the initiation of the cell therapy. For example,the subject may be administered a preconditioning agent at least 2 daysprior, such as at least 3, 4, 5, 6, or 7 days prior, to the initiationof the cell therapy. In some embodiments, the subject is administered apreconditioning agent no more than 7 days prior, such as no more than 6,5, 4, 3, or 2 days prior, to the initiation of the cell therapy.

In some embodiments, the subject is preconditioned with cyclophosphamideat a dose between or between about 20 mg/kg and 100 mg/kg, such asbetween or between about 40 mg/kg and 80 mg/kg. In some aspects, thesubject is preconditioned with or with about 60 mg/kg ofcyclophosphamide. In some embodiments, the cyclophosphamide can beadministered in a single dose or can be administered in a plurality ofdoses, such as given daily, every other day or every three days. In someembodiments, the cyclophosphamide is administered once daily for one ortwo days. In some embodiments, where the lymphodepleting agent comprisescyclophosphamide, the subject is administered cyclophosphamide at a dosebetween or between about 100 mg/m² and 500 mg/m², such as between orbetween about 200 mg/m² and 400 mg/m², or 250 mg/m² and 350 mg/m²,inclusive. In some instances, the subject is administered about 300mg/m² of cyclophosphamide. In some embodiments, the cyclophosphamide canbe administered in a single dose or can be administered in a pluralityof doses, such as given daily, every other day or every three days. Insome embodiments, cyclophosphamide is administered daily, such as for1-5 days, for example, for 3 to 5 days. In some instances, the subjectis administered about 300 mg/m² of cyclophosphamide, daily for 3 days,prior to initiation of the cell therapy.

In some embodiments, where the lymphodepleting agent comprisesfludarabine, the subject is administered fludarabine at a dose betweenor between about 1 mg/m² and 100 mg/m², such as between or between about10 mg/m² and 75 mg/m², 15 mg/m² and 50 mg/m², 20 mg/m² and 40 mg/m², or24 mg/m² and 35 mg/m², inclusive. In some instances, the subject isadministered about 30 mg/m² of fludarabine. In some embodiments, thefludarabine can be administered in a single dose or can be administeredin a plurality of doses, such as given daily, every other day or everythree days. In some embodiments, fludarabine is administered daily, suchas for 1-5 days, for example, for 3 to 5 days. In some instances, thesubject is administered about 30 mg/m² of fludarabine, daily for 3 days,prior to initiation of the cell therapy.

In some embodiments, the lymphodepleting agent comprises a combinationof agents, such as a combination of cyclophosphamide and fludarabine.Thus, the combination of agents may include cyclophosphamide at any doseor administration schedule, such as those described above, andfludarabine at any dose or administration schedule, such as thosedescribed above. For example, in some aspects, the subject isadministered 60 mg/kg (˜2 g/m²) of cyclophosphamide and 3 to 5 doses of25 mg/m² fludarabine prior to the first or subsequent dose.

Following administration of the cells, the biological activity of theengineered cell populations in some embodiments is measured, e.g., byany of a number of known methods. Parameters to assess include specificbinding of an engineered or natural T cell or other immune cell toantigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flowcytometry. In certain embodiments, the ability of the engineered cellsto destroy target cells can be measured using any suitable knownmethods, such as cytotoxicity assays described in, for example,Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Hermanet al. J. Immunological Methods, 285(1): 25-40 (2004). In certainembodiments, the biological activity of the cells is measured byassaying expression and/or secretion of one or more cytokines, such asCD107a, IFNγ, IL-2, and TNF. In some aspects the biological activity ismeasured by assessing clinical outcome, such as reduction in tumorburden or load.

In certain embodiments, the engineered cells are further modified in anynumber of ways, such that their therapeutic or prophylactic efficacy isincreased. For example, the engineered CAR or TCR expressed by thepopulation can be conjugated either directly or indirectly through alinker to a targeting moiety. The practice of conjugating compounds,e.g., the CAR or TCR, to targeting moieties is known. See, for instance,Wadwa et al., J. Drug Targeting 3: 1 1 1 (1995), and U.S. Pat. No.5,087,616. In some embodiments, the cells are administered as part of acombination treatment, such as simultaneously with or sequentially with,in any order, another therapeutic intervention, such as an antibody orengineered cell or receptor or agent, such as a cytotoxic or therapeuticagent. The cells in some embodiments are co-administered with one ormore additional therapeutic agents or in connection with anothertherapeutic intervention, either simultaneously or sequentially in anyorder. In some contexts, the cells are co-administered with anothertherapy sufficiently close in time such that the cell populationsenhance the effect of one or more additional therapeutic agents, or viceversa. In some embodiments, the cells are administered prior to the oneor more additional therapeutic agents. In some embodiments, the cellsare administered after the one or more additional therapeutic agents. Insome embodiments, the one or more additional agent includes a cytokine,such as IL-2, for example, to enhance persistence.

B. Dosing

In some embodiments, a dose of cells is administered to subjects inaccord with the provided methods, and/or with the provided articles ofmanufacture or compositions. In some embodiments, the size or timing ofthe doses is determined as a function of the particular disease orcondition in the subject. In some cases, the size or timing of the dosesfor a particular disease in view of the provided description may beempirically determined.

In some embodiments, the dose of cells comprises between at or about2×10⁵ of the cells/kg and at or about 2×10⁶ of the cells/kg, such asbetween at or about 4×10⁵ of the cells/kg and at or about 1×10⁶ of thecells/kg or between at or about 6×10⁵ of the cells/kg and at or about8×10⁵ of the cells/kg. In some embodiments, the dose of cells comprisesno more than 2×10⁵ of the cells (e.g. antigen-expressing, such asCAR-expressing cells) per kilogram body weight of the subject(cells/kg), such as no more than at or about 3×10⁵ cells/kg, no morethan at or about 4×10⁵ cells/kg, no more than at or about 5×10⁵cells/kg, no more than at or about 6×10⁵ cells/kg, no more than at orabout 7×10⁵ cells/kg, no more than at or about 8×10⁵ cells/kg, no morethan at or about 9×10⁵ cells/kg, no more than at or about 1×10⁶cells/kg, or no more than at or about 2×10⁶ cells/kg. In someembodiments, the dose of cells comprises at least or at least about orat or about 2×10⁵ of the cells (e.g. antigen-expressing, such asCAR-expressing cells) per kilogram body weight of the subject(cells/kg), such as at least or at least about or at or about 3×10⁵cells/kg, at least or at least about or at or about 4×10⁵ cells/kg, atleast or at least about or at or about 5×10⁵ cells/kg, at least or atleast about or at or about 6×10⁵ cells/kg, at least or at least about orat or about 7×10⁵ cells/kg, at least or at least about or at or about8×10⁵ cells/kg, at least or at least about or at or about 9×10⁵cells/kg, at least or at least about or at or about 1×10⁶ cells/kg, orat least or at least about or at or about 2×10⁶ cells/kg.

In certain embodiments, the cells, or individual populations ofsub-types of cells, are administered to the subject at a range of aboutone million to about 100 billion cells and/or that amount of cells perkilogram of body weight, such as, e.g., 1 million to about 50 billioncells (e.g., about 5 million cells, about 25 million cells, about 500million cells, about 1 billion cells, about 5 billion cells, about 20billion cells, about 30 billion cells, about 40 billion cells, or arange defined by any two of the foregoing values), such as about 10million to about 100 billion cells (e.g., about 20 million cells, about30 million cells, about 40 million cells, about 60 million cells, about70 million cells, about 80 million cells, about 90 million cells, about10 billion cells, about 25 billion cells, about 50 billion cells, about75 billion cells, about 90 billion cells, or a range defined by any twoof the foregoing values), and in some cases about 100 million cells toabout 50 billion cells (e.g., about 120 million cells, about 250 millioncells, about 350 million cells, about 450 million cells, about 650million cells, about 800 million cells, about 900 million cells, about 3billion cells, about 30 billion cells, about 45 billion cells) or anyvalue in between these ranges and/or per kilogram of body weight.Dosages may vary depending on attributes particular to the disease ordisorder and/or patient and/or other treatments.

In some embodiments, the dose of cells is a flat dose of cells or fixeddose of cells such that the dose of cells is not tied to or based on thebody surface area or weight of a subject.

In some embodiments, the dose of genetically engineered cells comprisesfrom or from about 1×10⁵ to 5×10⁸ total CAR-expressing T cells, 1×10⁵ to2.5×10⁸ total CAR-expressing T cells, 1×10⁵ to 1×10⁸ totalCAR-expressing T cells, 1×10⁵ to 5×10⁷ total CAR-expressing T cells,1×10⁵ to 2.5×10⁷ total CAR-expressing T cells, 1×10⁵ to 1×10⁷ totalCAR-expressing T cells, 1×10⁵ to 5×10⁶ total CAR-expressing T cells,1×10⁵ to 2.5×10⁶ total CAR-expressing T cells, 1×10⁵ to 1×10⁶ totalCAR-expressing T cells, 1×10⁶ to 5×10⁸ total CAR-expressing T cells,1×10⁶ to 2.5×10⁸ total CAR-expressing T cells, 1×10⁶ to 1×10⁸ totalCAR-expressing T cells, 1×10⁶ to 5×10⁷ total CAR-expressing T cells,1×10⁶ to 2.5×10⁷ total CAR-expressing T cells, 1×10⁶ to 1×10⁷ totalCAR-expressing T cells, 1×10⁶ to 5×10⁶ total CAR-expressing T cells,1×10⁶ to 2.5×10⁶ total CAR-expressing T cells, 2.5×10⁶ to 5×10⁸ totalCAR-expressing T cells, 2.5×10⁶ to 2.5×10⁸ total CAR-expressing T cells,2.5×10⁶ to 1×10⁸ total CAR-expressing T cells, 2.5×10⁶ to 5×10⁷ totalCAR-expressing T cells, 2.5×10⁶ to 2.5×10⁷ total CAR-expressing T cells,2.5×10⁶ to 1×10⁷ total CAR-expressing T cells, 2.5×10⁶ to 5×10⁶ totalCAR-expressing T cells, 5×10⁶ to 5×10⁸ total CAR-expressing T cells,5×10⁶ to 2.5×10⁸ total CAR-expressing T cells, 5×10⁶ to 1×10⁸ totalCAR-expressing T cells, 5×10⁶ to 5×10⁷ total CAR-expressing T cells,5×10⁶ to 2.5×10⁷ total CAR-expressing T cells, 5×10⁶ to 1×10⁷ totalCAR-expressing T cells, 1×10⁷ to 5×10⁸ total CAR-expressing T cells,1×10⁷ to 2.5×10⁸ total CAR-expressing T cells, 1×10⁷ to 1×10⁸ totalCAR-expressing T cells, 1×10⁷ to 5×10⁷ total CAR-expressing T cells,1×10⁷ to 2.5×10⁷ total CAR-expressing T cells, 2.5×10⁷ to 5×10⁸ totalCAR-expressing T cells, 2.5×10⁷ to 2.5×10⁸ total CAR-expressing T cells,2.5×10⁷ to 1×10⁸ total CAR-expressing T cells, 2.5×10⁷ to 5×10⁷ totalCAR-expressing T cells, 5×10⁷ to 5×10⁸ total CAR-expressing T cells,5×10⁷ to 2.5×10⁸ total CAR-expressing T cells, 5×10⁷ to 1×10⁸ totalCAR-expressing T cells, 1×10⁸ to 5×10⁸ total CAR-expressing T cells,1×10⁸ to 2.5×10⁸ total CAR-expressing T cells, or 2.5×10⁸ to 5×10⁸ totalCAR-expressing T cells.

In some embodiments, the dose of genetically engineered cells comprisesat least or at least about 1×10⁵ CAR-expressing cells, at least or atleast about 2.5×10⁵ CAR-expressing cells, at least or at least about5×10⁵ CAR-expressing cells, at least or at least about 1×10⁶CAR-expressing cells, at least or at least about 2.5×10⁶ CAR-expressingcells, at least or at least about 5×10⁶ CAR-expressing cells, at leastor at least about 1×10⁷ CAR-expressing cells, at least or at least about2.5×10⁷ CAR-expressing cells, at least or at least about 5×10⁷CAR-expressing cells, at least or at least about 1×10⁸ CAR-expressingcells, at least or at least about 2.5×10⁸ CAR-expressing cells, or atleast or at least about 5×10⁸ CAR-expressing cells.

In some embodiments, the cell therapy comprises administration of a dosecomprising a number of cell from or from about 1×10⁵ to 5×10⁸ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), from or from about 5×10⁵ to1×10⁷ total recombinant receptor-expressing cells, total T cells, ortotal peripheral blood mononuclear cells (PBMCs) or from or from about1×10⁶ to 1×10⁷ total recombinant receptor-expressing cells, total Tcells, or total peripheral blood mononuclear cells (PBMCs), eachinclusive. In some embodiments, the cell therapy comprisesadministration of a dose of cells comprising a number of cells at leastor at least about 1×10⁵ total recombinant receptor-expressing cells,total T cells, or total peripheral blood mononuclear cells (PBMCs), suchat least or at least 1×10⁶, at least or at least about 1×10⁷, at leastor at least about 1×10⁸ of such cells. In some embodiments, the numberis with reference to the total number of CD3⁺ or CD8⁺, in some casesalso recombinant receptor-expressing (e.g. CAR⁺) cells. In someembodiments, the cell therapy comprises administration of a dosecomprising a number of cell from or from about 1×10⁵ to 5×10⁸ CD3⁺ orCD8⁺ total T cells or CD3⁺ or CD8⁺ recombinant receptor-expressingcells, from or from about 5×10⁵ to 1×10⁷ CD3⁺ or CD8⁺ total T cells orCD3⁺ or CD8⁺ recombinant receptor-expressing cells, or from or fromabout 1×10⁶ to 1×10⁷ CD3⁺ or CD8⁺ total T cells or CD3⁺ or CD8⁺recombinant receptor-expressing cells, each inclusive. In someembodiments, the cell therapy comprises administration of a dosecomprising a number of cell from or from about 1×10⁵ to 5×10⁸ totalCD3⁺/CAR⁺ or CD8⁺/CAR⁺ cells, from or from about 5×10⁵ to 1×10⁷ totalCD3⁺/CAR⁺ or CD8⁺/CAR⁺ cells, or from or from about 1×10⁶ to 1×10⁷ totalCD3⁺/CAR⁺ or CD8⁺/CAR⁺ cells, each inclusive.

In some embodiments, the dose of T cells comprises: at or about 5×10⁷recombinant receptor-expressing T cells or at or about 2.5×10⁷recombinant receptor-expressing CD8⁺ T cells. In some embodiments, thedose of T cells comprises: at or about 1×10⁸ recombinantreceptor-expressing T cells or at or about 5×10⁷ recombinantreceptor-expressing CD8⁺ T cells. In some embodiments, the dose of Tcells comprises: at or about 1.5×10⁸ recombinant receptor-expressing Tcells or at or about 0.75×10⁸ recombinant receptor-expressing CD8⁺ Tcells.

In some embodiments, the T cells of the dose include CD4⁺ T cells, CD8⁺T cells or CD4⁺ and CD8⁺ T cells.

In some embodiments, for example, where the subject is human, the CD8⁺ Tcells of the dose, including in a dose including CD4⁺ and CD8⁺ T cells,includes between about 1×10⁶ and 5×10⁸ total recombinant receptor (e.g.,CAR)-expressing CD8⁺ cells, e.g., in the range of about 5×10⁶ to 1×10⁸such cells, such cells 1×10⁷, 2.5×10⁷, 5×10⁷, 7.5×10⁷, 1×10⁸, or 5×10⁸total such cells, or the range between any two of the foregoing values.In some embodiments, the patient is administered multiple doses, andeach of the doses or the total dose can be within any of the foregoingvalues. In some embodiments, the dose of cells comprises theadministration of from or from about 1×10⁷ to 0.75×10⁸ total recombinantreceptor-expressing CD8⁺ T cells, 1×10⁷ to 2.5×10⁷ total recombinantreceptor-expressing CD8⁺ T cells, from or from about 1×10⁷ to 0.75×10⁸total recombinant receptor-expressing CD8⁺ T cells, each inclusive. Insome embodiments, the dose of cells comprises the administration of orabout 1×10⁷, 2.5×10⁷, 5×10⁷ 7.5×10⁷, 1×10⁸, or 5×10⁸ total recombinantreceptor-expressing CD8⁺ T cells.

In some embodiments, for example, where the subject is a human, the doseincludes fewer than about 2×10⁸ 1×10⁸ total recombinant receptor (e.g.,CAR)-expressing cells, T cells, or peripheral blood mononuclear cells(PBMCs), e.g., in the range of about 1×10⁶ to 2×10⁸ or 1×10⁶ to 1×10⁸such cells, such as 2×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸ or 1.5×10⁸ totalsuch cells, or the range between any two of the foregoing values. Insome embodiments, where the subject is a human, the dose includesbetween about 1×10⁶ and 3×10⁸ total recombinant receptor (e.g.,CAR)-expressing cells, e.g., in the range of about 1×10⁷ to 2×10⁸ suchcells, such as 1×10⁷, 5×10⁷, 1×10⁸ or 1.5×10⁸ total such cells, or therange between any two of the foregoing values. In some embodiments, thepatient is administered multiple doses, and each of the doses or thetotal dose can be within any of the foregoing values. In someembodiments, the dose of cells comprises the administration of from orfrom about 1×10⁵ to 5×10⁸ total recombinant receptor-expressing T cellsor total T cells, 1×10⁵ to 1.5×10⁸ total recombinant receptor-expressingT cells or total T cells, 1×10⁵ to 1×10⁸ total recombinantreceptor-expressing T cells or total T cells, from or from about 5×10⁵to 1×10⁷ total recombinant receptor-expressing T cells or total T cells,or from or from about 1×10⁶ to 1×10⁷ total recombinantreceptor-expressing T cells or total T cells, each inclusive.

In some embodiments, the T cells of the dose include CD4⁺ T cells, CD8⁺T cells or CD4⁺ and CD8⁺ T cells.

In some embodiments, for example, where the subject is human, the CD8⁺ Tcells of the dose, including in a dose including CD4⁺ and CD8⁺ T cells,includes between about 1×10⁶ and 1×10⁸ total recombinant receptor (e.g.,CAR)-expressing CD8⁺ cells, e.g., in the range of about 5×10⁶ to 1×10⁸such cells, such cells 1×10⁷, 2.5×10⁷, 5×10⁷, 7.5×10⁷ or 1×10⁸ totalsuch cells, or the range between any two of the foregoing values. Insome embodiments, the patient is administered multiple doses, and eachof the doses or the total dose can be within any of the foregoingvalues. In some embodiments, the dose of cells comprises theadministration of from or from about 1×10⁷ to 0.75×10⁸ total recombinantreceptor-expressing CD8⁺ T cells, 1×10⁷ to 2.5×10⁷ total recombinantreceptor-expressing CD8⁺ T cells, from or from about 1×10⁷ to 0.75×10⁸total recombinant receptor-expressing CD8⁺ T cells, each inclusive. Insome embodiments, the dose of cells comprises the administration of orabout 1×10⁷, 2.5×10⁷, 5×10⁷ 7.5×10⁷ or 1×10⁸ total recombinantreceptor-expressing CD8⁺ T cells.

In some embodiments, the dose of cells, e.g., recombinantreceptor-expressing T cells, is administered to the subject as a singledose or is administered only one time within a period of two weeks, onemonth, three months, six months, 1 year or more.

In the context of adoptive cell therapy, administration of a given“dose” encompasses administration of the given amount or number of cellsas a single composition and/or single uninterrupted administration,e.g., as a single injection or continuous infusion, and also encompassesadministration of the given amount or number of cells as a split dose oras a plurality of compositions, provided in multiple individualcompositions or infusions, over a specified period of time, such as overno more than 3 days. Thus, in some contexts, the dose is a single orcontinuous administration of the specified number of cells, given orinitiated at a single point in time. In some contexts, however, the doseis administered in multiple injections or infusions over a period of nomore than three days, such as once a day for three days or for two daysor by multiple infusions over a single day period.

Thus, in some aspects, the cells of the dose are administered in asingle pharmaceutical composition. In some embodiments, the cells of thedose are administered in a plurality of compositions, collectivelycontaining the cells of the dose.

In some embodiments, the term “split dose” refers to a dose that issplit so that it is administered over more than one day. This type ofdosing is encompassed by the present methods and is considered to be asingle dose.

Thus, the dose of cells may be administered as a split dose, e.g., asplit dose administered over time. For example, in some embodiments, thedose may be administered to the subject over 2 days or over 3 days.Exemplary methods for split dosing include administering 25% of the doseon the first day and administering the remaining 75% of the dose on thesecond day. In other embodiments, 33% of the dose may be administered onthe first day and the remaining 67% administered on the second day. Insome aspects, 10% of the dose is administered on the first day, 30% ofthe dose is administered on the second day, and 60% of the dose isadministered on the third day. In some embodiments, the split dose isnot spread over more than 3 days.

In some embodiments, cells of the dose may be administered byadministration of a plurality of compositions or solutions, such as afirst and a second, optionally more, each containing some cells of thedose. In some aspects, the plurality of compositions, each containing adifferent population and/or sub-types of cells, are administeredseparately or independently, optionally within a certain period of time.For example, the populations or sub-types of cells can include CD8⁺ andCD4⁺ T cells, respectively, and/or CD8⁺- and CD4⁺-enriched populations,respectively, e.g., CD4⁺ and/or CD8⁺ T cells each individually includingcells genetically engineered to express the recombinant receptor. Insome embodiments, the administration of the dose comprisesadministration of a first composition comprising a dose of CD8⁺ T cellsor a dose of CD4⁺ T cells and administration of a second compositioncomprising the other of the dose of CD4⁺ T cells and the CD8⁺ T cells.

In some embodiments, the administration of the composition or dose,e.g., administration of the plurality of cell compositions, involvesadministration of the cell compositions separately. In some aspects, theseparate administrations are carried out simultaneously, orsequentially, in any order. In some embodiments, the dose comprises afirst composition and a second composition, and the first compositionand second composition are administered 0 to 12 hours apart, 0 to 6hours apart or 0 to 2 hours apart. In some embodiments, the initiationof administration of the first composition and the initiation ofadministration of the second composition are carried out no more than 2hours, no more than 1 hour, or no more than 30 minutes apart, no morethan 15 minutes, no more than 10 minutes or no more than 5 minutesapart. In some embodiments, the initiation and/or completion ofadministration of the first composition and the completion and/orinitiation of administration of the second composition are carried outno more than 2 hours, no more than 1 hour, or no more than 30 minutesapart, no more than 15 minutes, no more than 10 minutes or no more than5 minutes apart.

In some composition, the first composition, e.g., first composition ofthe dose, comprises CD4⁺ T cells. In some composition, the firstcomposition, e.g., first composition of the dose, comprises CD8⁺ Tcells. In some embodiments, the first composition is administered priorto the second composition.

In some embodiments, the dose or composition of cells includes a definedor target ratio of CD4⁺ cells expressing a recombinant receptor to CD8⁺cells expressing a recombinant receptor and/or of CD4⁺ cells to CD8⁺cells, which ratio optionally is approximately 1:1 or is betweenapproximately 1:3 and approximately 3:1, such as approximately 1:1. Insome aspects, the administration of a composition or dose with thetarget or desired ratio of different cell populations (such as CD4⁺:CD8⁺ratio or CAR⁺CD4⁺:CAR⁺CD8⁺ ratio, e.g., 1:1) involves the administrationof a cell composition containing one of the populations and thenadministration of a separate cell composition comprising the other ofthe populations, where the administration is at or approximately at thetarget or desired ratio. In some aspects, administration of a dose orcomposition of cells at a defined ratio leads to improved expansion,persistence and/or antitumor activity of the T cell therapy.

In some embodiments, the subject receives multiple doses, e.g., two ormore doses or multiple consecutive doses, of the cells. In someembodiments, two doses are administered to a subject. In someembodiments, the subject receives the consecutive dose, e.g., seconddose, is administered approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose. In someembodiments, multiple consecutive doses are administered following thefirst dose, such that an additional dose or doses are administeredfollowing administration of the consecutive dose. In some aspects, thenumber of cells administered to the subject in the additional dose isthe same as or similar to the first dose and/or consecutive dose. Insome embodiments, the additional dose or doses are larger than priordoses.

In some aspects, the size of the first and/or consecutive dose isdetermined based on one or more criteria such as response of the subjectto prior treatment, e.g. chemotherapy, disease burden in the subject,such as tumor load, bulk, size, or degree, extent, or type ofmetastasis, stage, and/or likelihood or incidence of the subjectdeveloping toxic outcomes, e.g., CRS, macrophage activation syndrome,tumor lysis syndrome, neurotoxicity, and/or a host immune responseagainst the cells and/or recombinant receptors being administered.

In some aspects, the time between the administration of the first doseand the administration of the consecutive dose is about 9 to about 35days, about 14 to about 28 days, or 15 to 27 days. In some embodiments,the administration of the consecutive dose is at a time point more thanabout 14 days after and less than about 28 days after the administrationof the first dose. In some aspects, the time between the first andconsecutive dose is about 21 days. In some embodiments, an additionaldose or doses, e.g. consecutive doses, are administered followingadministration of the consecutive dose. In some aspects, the additionalconsecutive dose or doses are administered at least about 14 and lessthan about 28 days following administration of a prior dose. In someembodiments, the additional dose is administered less than about 14 daysfollowing the prior dose, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, or13 days after the prior dose. In some embodiments, no dose isadministered less than about 14 days following the prior dose and/or nodose is administered more than about 28 days after the prior dose.

In some embodiments, the dose of cells, e.g., recombinantreceptor-expressing cells, comprises two doses (e.g., a double dose),comprising a first dose of the T cells and a consecutive dose of the Tcells, wherein one or both of the first dose and the second dosecomprises administration of the split dose of T cells.

In some embodiments, the dose of cells is generally large enough to beeffective in reducing disease burden.

In some embodiments, the cells are administered at a desired dosage,which in some aspects includes a desired dose or number of cells or celltype(s) and/or a desired ratio of cell types. Thus, the dosage of cellsin some embodiments is based on a total number of cells (or number perkg body weight) and a desired ratio of the individual populations orsub-types, such as the CD4⁺ to CD8⁺ ratio. In some embodiments, thedosage of cells is based on a desired total number (or number per kg ofbody weight) of cells in the individual populations or of individualcell types. In some embodiments, the dosage is based on a combination ofsuch features, such as a desired number of total cells, desired ratio,and desired total number of cells in the individual populations.

In some embodiments, the populations or sub-types of cells, such as CD8⁺and CD4⁺ T cells, are administered at or within a tolerated differenceof a desired dose of total cells, such as a desired dose of T cells. Insome aspects, the desired dose is a desired number of cells or a desirednumber of cells per unit of body weight of the subject to whom the cellsare administered, e.g., cells/kg. In some aspects, the desired dose isat or above a minimum number of cells or minimum number of cells perunit of body weight. In some aspects, among the total cells,administered at the desired dose, the individual populations orsub-types are present at or near a desired output ratio (such as CD4⁺ toCD8⁺ ratio), e.g., within a certain tolerated difference or error ofsuch a ratio.

In some embodiments, the cells are administered at or within a tolerateddifference of a desired dose of one or more of the individualpopulations or sub-types of cells, such as a desired dose of CD4⁺ cellsand/or a desired dose of CD8⁺ cells. In some aspects, the desired doseis a desired number of cells of the sub-type or population, or a desirednumber of such cells per unit of body weight of the subject to whom thecells are administered, e.g., cells/kg. In some aspects, the desireddose is at or above a minimum number of cells of the population orsub-type, or minimum number of cells of the population or sub-type perunit of body weight.

Thus, in some embodiments, the dosage is based on a desired fixed doseof total cells and a desired ratio, and/or based on a desired fixed doseof one or more, e.g., each, of the individual sub-types orsub-populations. Thus, in some embodiments, the dosage is based on adesired fixed or minimum dose of T cells and a desired ratio of CD4⁺ toCD8⁺ cells, and/or is based on a desired fixed or minimum dose of CD4⁺and/or CD8⁺ cells.

In some embodiments, the cells are administered at or within a toleratedrange of a desired output ratio of multiple cell populations orsub-types, such as CD4⁺ and CD8⁺ cells or sub-types. In some aspects,the desired ratio can be a specific ratio or can be a range of ratios.for example, in some embodiments, the desired ratio (e.g., ratio of CD4⁺to CD8⁺ cells) is between at or about 5:1 and at or about 5:1 (orgreater than about 1:5 and less than about 5:1), or between at or about1:3 and at or about 3:1 (or greater than about 1:3 and less than about3:1), such as between at or about 2:1 and at or about 1:5 (or greaterthan about 1:5 and less than about 2:1, such as at or about 5:1, 4.5:1,4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9:1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In someaspects, the tolerated difference is within about 1%, about 2%, about3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50% of the desired ratio,including any value in between these ranges.

In particular embodiments, the numbers and/or concentrations of cellsrefer to the number of recombinant receptor (e.g., CAR)-expressingcells. In other embodiments, the numbers and/or concentrations of cellsrefer to the number or concentration of all cells, T cells, orperipheral blood mononuclear cells (PBMCs) administered.

In some aspects, the size of the dose is determined based on one or morecriteria such as response of the subject to prior treatment, e.g.chemotherapy, disease burden in the subject, such as tumor load, bulk,size, or degree, extent, or type of metastasis, stage, and/or likelihoodor incidence of the subject developing toxic outcomes, e.g., CRS,macrophage activation syndrome, tumor lysis syndrome, neurotoxicity,and/or a host immune response against the cells and/or recombinantreceptors being administered.

In some embodiments, the methods also include administering one or moreadditional doses of cells expressing a chimeric antigen receptor (CAR)and/or lymphodepleting therapy, and/or one or more steps of the methodsare repeated. In some embodiments, the one or more additional dose isthe same as the initial dose. In some embodiments, the one or moreadditional dose is different from the initial dose, e.g., higher, suchas 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or10-fold or more higher than the initial dose, or lower, such as e.g.,higher, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold or 10-fold or more lower than the initial dose. In someembodiments, administration of one or more additional doses isdetermined based on response of the subject to the initial treatment orany prior treatment, disease burden in the subject, such as tumor load,bulk, size, or degree, extent, or type of metastasis, stage, and/orlikelihood or incidence of the subject developing toxic outcomes, e.g.,CRS, macrophage activation syndrome, tumor lysis syndrome,neurotoxicity, and/or a host immune response against the cells and/orrecombinant receptors being administered.

C. Response, Efficacy and Survival

In some embodiments, the administration effectively treats the subjectdespite the subject having become resistant to another therapy. In someembodiments, at least 30%, at least 35%, at least 40% or at least 50% ofsubjects treated according to the method achieve complete remission(CR); and/or at least about 40%, at least about 50%, at least about 60%or at least about 70% of the subjects treated according to the methodachieve an objective response (OR). In some embodiments, at least orabout at least 50% of subjects, at least or about at least 60% of thesubjects, at least or about at least 70% of the subjects, at least orabout at least 80% of the subjects or at least or about at least 90% ofthe subjects treated according to the method achieve CR and/or achievean objective response (OR). In some embodiments, criteria assessed foreffective treatment includes overall response rate (ORR; also known insome cases as objective response rate), complete response (CR; alsoknown in some cases as complete response), duration of response (DOR)progression-free survival (PFS), and/or overall survival (OS).

In some embodiments, at least 40% or at least 50% of subjects treatedaccording to the methods provided herein achieve complete remission (CR;also known in some cases as complete response), exhibit progression-freesurvival (PFS) and/or overall survival (OS) of greater than at or about3 months, 6 months or 12 months or greater than 13 months orapproximately 14 months; on average, subjects treated according to themethod exhibit a median PFS or OS of greater than at or about 6 months,12 months, or 18 months; and/or the subject exhibits PFS or OS followingtherapy for at least at or about 6, 12, 18 or more months or longer.

In some aspects, response rates in subjects, such as subjects with NHL,are based on the Lugano criteria. (Cheson et al., (2014) JCO32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin Clin Oncol 4(1):5). In some aspects, response assessmentutilizes any of clinical, hematologic, and/or molecular methods. In someaspects, response assessed using the Lugano criteria involves the use ofpositron emission tomography (PET)-computed tomography (CT) and/or CT asappropriate. PET-CT evaluations may further comprise the use offluorodeoxyglucose (FDG) for FDG-avid lymphomas. In some aspects, wherePET-CT will be used to assess response in FDG-avid histologies, a5-point scale may be used. In some respects, the 5-point scale comprisesthe following criteria: 1, no uptake above background; 2, uptake≤mediastinum; 3, uptake >mediastinum but ≤liver; 4, uptakemoderately >liver; 5, uptake markedly higher than liver and/or newlesions; X, new areas of uptake unlikely to be related to lymphoma.

In some aspects, a complete response as described using the Luganocriteria involves a complete metabolic response and a completeradiologic response at various measureable sites. In some aspects, thesesites include lymph nodes and extralymphatic sites, wherein a CR isdescribed as a score of 1, 2, or 3 with or without a residual mass onthe 5-point scale, when PET-CT is used. In some aspects, in Waldeyer'sring or extranodal sites with high physiologic uptake or with activationwithin spleen or marrow (e.g., with chemotherapy or myeloidcolony-stimulating factors), uptake may be greater than normalmediastinum and/or liver. In this circumstance, complete metabolicresponse may be inferred if uptake at sites of initial involvement is nogreater than surrounding normal tissue even if the tissue has highphysiologic uptake. In some aspects, response is assessed in the lymphnodes using CT, wherein a CR is described as no extralymphatic sites ofdisease and target nodes/nodal masses must regress to ≤1.5 cm in longesttransverse diameter of a lesion (LDi). Further sites of assessmentinclude the bone marrow wherein PET-CT-based assessment should indicatea lack of evidence of FDG-avid disease in marrow and a CT-basedassessment should indicate a normal morphology, which if indeterminateshould be IHC negative. Further sites may include assessment of organenlargement, which should regress to normal. In some aspects,nonmeasured lesions and new lesions are assessed, which in the case ofCR should be absent (Cheson et al., (2014) JCO 32(27):3059-3067; Johnsonet al., (2015) Radiology 2:323-338; Cheson, B. D. (2015) Chin Clin Oncol4(1):5).

In some aspects, a partial response (PR; also known in some cases aspartial remission) as described using the Lugano criteria involves apartial metabolic and/or radiological response at various measureablesites. In some aspects, these sites include lymph nodes andextralymphatic sites, wherein a PR is described as a score of 4 or 5with reduced uptake compared with baseline and residual mass(es) of anysize, when PET-CT is used. At interim, such findings can indicateresponding disease. At the end of treatment, such findings can indicateresidual disease. In some aspects, response is assessed in the lymphnodes using CT, wherein a PR is described as ≥50% decrease in SPD of upto 6 target measureable nodes and extranodal sites. If a lesion is toosmall to measure on CT, 5 mm×5 mm is assigned as the default value; ifthe lesion is no longer visible, the value is 0 mm×0 mm; for a node >5mm×5 mm, but smaller than normal, actual measurements are used forcalculation. Further sites of assessment include the bone marrow whereinPET-CT-based assessment should indicate residual uptake higher thanuptake in normal marrow but reduced compared with baseline (diffuseuptake compatible with reactive changes from chemotherapy allowed). Insome aspects, if there are persistent focal changes in the marrow in thecontext of a nodal response, consideration should be given to furtherevaluation with MRI or biopsy, or an interval scan. In some aspects,further sites may include assessment of organ enlargement, where thespleen must have regressed by >50% in length beyond normal. In someaspects, nonmeasured lesions and new lesions are assessed, which in thecase of PR should be absent/normal, regressed, but no increase. Noresponse/stable disease (SD) or progressive disease (PD) can also bemeasured using PET-CT and/or CT based assessments. (Cheson et al.,(2014) JCO 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338;Cheson, B. D. (2015) Chin Clin Oncol 4(1):5).

In some respects, progression-free survival (PFS) is described as thelength of time during and after the treatment of a disease, such ascancer, that a subject lives with the disease but it does not get worse.In some aspects, objective response (OR) is described as a measurableresponse. In some aspects, objective response rate (ORR; also known insome cases as overall response rate) is described as the proportion ofpatients who achieved CR or PR. In some aspects, overall survival (OS)is described as the length of time from either the date of diagnosis orthe start of treatment for a disease, such as cancer, that subjectsdiagnosed with the disease are still alive. In some aspects, event-freesurvival (EFS) is described as the length of time after treatment for acancer ends that the subject remains free of certain complications orevents that the treatment was intended to prevent or delay. These eventsmay include the return of the cancer or the onset of certain symptoms,such as bone pain from cancer that has spread to the bone, or death.

In some embodiments, the measure of duration of response (DOR) includesthe time from documentation of tumor response to disease progression. Insome embodiments, the parameter for assessing response can includedurable response, e.g., response that persists after a period of timefrom initiation of therapy. In some embodiments, durable response isindicated by the response rate at approximately 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 18 or 24 months after initiation of therapy. In someembodiments, the response is durable for greater than 3 months orgreater than 6 months.

In some aspects, the RECIST criteria is used to determine objectivetumor response; in some aspects, in solid tumors. (Eisenhauer et al.,European Journal of Cancer 45 (2009) 228-247.) In some aspects, theRECIST criteria is used to determine objective tumor response for targetlesions. In some respects, a complete response as determined usingRECIST criteria is described as the disappearance of all target lesionsand any pathological lymph nodes (whether target or non-target) musthave reduction in short axis to <10 mm. In other aspects, a partialresponse as determined using RECIST criteria is described as at least a30% decrease in the sum of diameters of target lesions, taking asreference the baseline sum diameters. In other aspects, progressivedisease (PD) is described as at least a 20% increase in the sum ofdiameters of target lesions, taking as reference the smallest sum onstudy (this includes the baseline sum if that is the smallest on study).In addition to the relative increase of 20%, the sum must alsodemonstrate an absolute increase of at least 5 mm (in some aspects theappearance of one or more new lesions is also considered progression).In other aspects, stable disease (SD) is described as neither sufficientshrinkage to qualify for PR nor sufficient increase to qualify for PD,taking as reference the smallest sum diameters while on study.

In some aspects, the administration in accord with the provided methods,and/or with the provided articles of manufacture or compositions,generally reduces or prevents the expansion or burden of the disease orcondition in the subject. For example, where the disease or condition isa tumor, the methods generally reduce tumor size, bulk, metastasis,percentage of blasts in the bone marrow or molecularly detectable cancerand/or improve prognosis or survival or other symptom associated withtumor burden.

Disease burden can encompass a total number of cells of the disease inthe subject or in an organ, tissue, or bodily fluid of the subject, suchas the organ or tissue of the tumor or another location, e.g., whichwould indicate metastasis. For example, tumor cells may be detectedand/or quantified in the blood or bone marrow in the context of certainhematological malignancies. Disease burden can include, in someembodiments, the mass of a tumor, the number or extent of metastasesand/or the percentage of blast cells present in the bone marrow.

In some embodiments, a subject has leukemia. The extent of diseaseburden can be determined by assessment of residual leukemia in blood orbone marrow.

In some aspects, response rates in subjects, such as subjects with CLL,are based on the International Workshop on Chronic Lymphocytic Leukemia(IWCLL) response criteria (Hallek, et al., Blood 2008, Jun. 15; 111(12):5446-5456). In some aspects, these criteria are described as follows:complete remission (CR; also known in some cases as complete response),which in some aspects requires the absence of peripheral blood clonallymphocytes by immunophenotyping, absence of lymphadenopathy, absence ofhepatomegaly or splenomegaly, absence of constitutional symptoms andsatisfactory blood counts; complete remission with incomplete marrowrecovery (CRi), which in some aspects is described as CR above, butwithout normal blood counts; partial remission (PR; also known in somecases as partial response), which in some aspects is described as ≥50%fall in lymphocyte count, ≥50% reduction in lymphadenopathy or ≥50%reduction in liver or spleen, together with improvement in peripheralblood counts; progressive disease (PD), which in some aspects isdescribed as ≥50% rise in lymphocyte count to >5×10⁹/L, ≥50% increase inlymphadenopathy, ≥50% increase in liver or spleen size, Richter'stransformation, or new cytopenias due to CLL; and stable disease, whichin some aspects is described as not meeting criteria for CR, CRi, PR orPD.

In some embodiments, the subjects exhibits a CR or OR if, within 1 monthof the administration of the dose of cells, lymph nodes in the subjectare less than at or about 20 mm in size, less than at or about 10 mm insize or less than at or about 10 mm in size.

In some embodiments, an index clone of the CLL is not detected in thebone marrow of the subject (or in the bone marrow of greater than 50%,60%, 70%, 80%, 90% or more of the subjects treated according to themethods. In some embodiments, an index clone of the CLL is assessed byIgH deep sequencing. In some embodiments, the index clone is notdetected at a time that is at or about or at least at or about 1, 2, 3,4, 5, 6, 12, 18 or 24 months following the administration of the cells.

In some embodiments, a subject exhibits morphologic disease if there aregreater than or equal to 5% blasts in the bone marrow, for example, asdetected by light microscopy, such as greater than or equal to 10%blasts in the bone marrow, greater than or equal to 20% blasts in thebone marrow, greater than or equal to 30% blasts in the bone marrow,greater than or equal to 40% blasts in the bone marrow or greater thanor equal to 50% blasts in the bone marrow. In some embodiments, asubject exhibits complete or clinical remission if there are less than5% blasts in the bone marrow.

In some embodiments, a subject has leukemia. The extent of diseaseburden can be determined by assessment of residual leukemia in blood orbone marrow.

In some embodiments, a subject exhibits morphologic disease if there aregreater than or equal to 5% blasts in the bone marrow, for example, asdetected by light microscopy, such as greater than or equal to 10%blasts in the bone marrow, greater than or equal to 20% blasts in thebone marrow, greater than or equal to 30% blasts in the bone marrow,greater than or equal to 40% blasts in the bone marrow or greater thanor equal to 50% blasts in the bone marrow. In some embodiments, asubject exhibits complete or clinical remission if there are less than5% blasts in the bone marrow.

In some embodiments, a subject may exhibit complete remission, but asmall proportion of morphologically undetectable (by light microscopytechniques) residual leukemic cells are present. A subject is said toexhibit minimum residual disease (MRD) if the subject exhibits less than5% blasts in the bone marrow and exhibits molecularly detectable cancer.In some embodiments, molecularly detectable cancer can be assessed usingany of a variety of molecular techniques that permit sensitive detectionof a small number of cells. In some aspects, such techniques include PCRassays, which can determine unique Ig/T-cell receptor generearrangements or fusion transcripts produced by chromosometranslocations. In some embodiments, flow cytometry can be used toidentify cancer cell based on leukemia-specific immunophenotypes. Insome embodiments, molecular detection of cancer can detect as few as 1leukemia cell in 100,000 normal cells. In some embodiments, a subjectexhibits MRD that is molecularly detectable if at least or greater than1 leukemia cell in 100,000 cells is detected, such as by PCR or flowcytometry. In some embodiments, the disease burden of a subject ismolecularly undetectable or MRD⁻, such that, in some cases, no leukemiacells are able to be detected in the subject using PCR or flow cytometrytechniques.

In some embodiments, an index clone of the leukemia, e.g. CLL, is notdetected in the bone marrow of the subject (or in the bone marrow ofgreater than 50%, 60%, 70%, 80%, 90% or more of the subjects treatedaccording to the methods. In some embodiments, an index clone of theleukemia, e.g. CLL, is assessed by IGH deep sequencing. In someembodiments, the index clone is not detected at a time that is at orabout or at least at or about 1, 2, 3, 4, 5, 6, 12, 18 or 24 monthsfollowing the administration of the cells.

In some aspects MRD is detected by flow cytometry. Flow cytometry can beused to monitor bone marrow and peripheral blood samples for cancercells. In particular aspects, flow cytometry is used to detect ormonitor the presence of cancer cells in bone marrow. In some aspects,multiparameter immunological detection by flow cytometry is used todetect cancer cells (see for example, Coustan-Smith et al., (1998)Lancet 351:550-554). In some aspects, multiparameter immunologicaldetection by mass cytometry is used to detect cancer cells. In someexamples, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30,35, 40, 45 or 50 parameters can be used to detect cancer cells. Theantigens used for detection are selected based on the cancer beingdetected (Foon and Todd (1986) Blood 68:1-31).

In some examples, bone marrow is harvested by bone marrow aspirates orbone marrow biopsies, and lymphocytes are isolated for analysis.Monoclonal and/or polyclonal antibodies conjugated to a fluorochrome(e.g., fluorescein isothiocyanate (FITC), phycoerythrin, peridininchlorophyll protein, or biotin) can be used to detect epitopes, such asterminal deoxynucleotidyl transferase (TdT), CD3, CD10, CD11c, CD13,CD14, CD33, CD19, CD20, CD21, CD22, CD23, CD34, CD45, CD56, CD79b, IgM,and/or KORSA3544, on isolated lymphocytes. Labeled cells can then bedetected using flow cytometry, such as multiparameter flow cytometry, ormass cytometry, to detect multiple epitopes.

Lymphoid cells can be identified and gated based on a light-scatter dotplot and then secondarily gated to identify cell populations expressingthe immunophenotypic features of interest. Exemplary epitopes are setforth in Table 2 below. Other immunologic classification of leukemiasand lymphomas are provided by Foon and Todd (Blood (1986) 68(1): 1-31).In some aspects, flow cytometric assessment of MRD can be achieved byquantifying live lymphocytes bearing one or more CLL immunophenotypes(e.g., low forward/side scatter; CD3^(neg); CD5⁺; CD14^(neg); CD19⁺;CD23⁺; CD45⁺; CD56^(neg)).

TABLE 2 Exemplary Immnunophenotype and Cytogentics CharacteristicsDisease Immunophenotype Cytogenetics Chronic Pan-B+; CD5+; Trisomy12Lymphocytic CD23+; CD79b/CD22 del(13)(q14.3) Leukemia (CLL) weak; FMC7−;sIg del 11q22-q23 weak del 17p13 (p53) t(11; 14)(q13; q32) BCL1/IgHrearrangement t(14; 19)(q32; q13) IgH deletion (14q32) del(6q) +8q24 +3+18 del 6q21 Small lymphocytic Pan-B+; CD5+; del(6)(q21-23) lymphoma(SLL) CD23+; CD10−; sIgM+ faint Lymphoplasmacytic Pan-B+; CD5−; t(9;14)(p13; q32) PAX5/IgH lymphoma CD10−; cyIgM+ Follicle centre cellPan-B+; CD10+/−; t(14; 18)(q32; q21)/BCL2 Rearr lymphoma CD5−; sIg+Diffuse large cell CD19+; CD22+; t(14; 18) and p53 mutations lymphomaCD10−/+; SIg+ t(3; V)(q27; V)/BCL6 Rearr variants c-MYC Rearr Burkitt'slymphoma Pan-B+; TdT−; t(8; 14)(q24; q32) or variants/c-MYC Rearr CD10+;CD5−; sIgM+ Burkitt-like Pan-B+; TdT−; CD10−/+ t(8; 14) or variantslymphoma CD5−; sIg+ t(8; 14)+ t(14; 18) Mantle cell Pan-B+; CD5+; t(11;14)(q13; q32)/BCL1 Rearr lymphoma CD23−; CD10−/+; sIgM+ bright Marginalzone B-cell pan-B+; CD5−/+; t(11; 18)(q21; q21)/PI2/MLT fusion: Extra-lymphoma CD10−; CD23−; nodal low-grade MALT lymphoma; (MZBCL) CD11c+/−;cyIg+ indolent disease (40% of the cells), t(1; 14)(p21; q32):Extra-nodal MALT sIgM+ bright; sIgD− lymphoma del(7)(q22-31): SplenicMZBCL/+3q: Nodal, extra-nodal and splenic MZBCL +: positive in >90% ofthe cases +/−: positive in more than 50% of the cases −/+: positive inless than 50% of cases positive in <10% of the cases Pan-B markers:e.g., CD19, CD20, CD79a sIG: surface immunoglobulins cylg: cytoplasmicimmunoglobulins

In some aspects, deep sequencing of the immunoglobulin heavy chain (IGH)locus of harvested B cells can be used to detect minimal residualdisease (MRD). Clonal presence of a particular IgG rearrangement canprovide a marker to detect the presence of B cell malignancies, such asCLL or NHL and/or residual presence of malignant cells thereof. In someaspects cells such as a population containing or suspected of containingB cells are harvested and isolated from blood. In some aspects, cellsare harvested and isolated from bone marrow, e.g., from bone marrowaspirates or bone marrow biopsies and/or from other biological samples.In some aspects, polymerase chain reaction (PCR) amplification of thecomplementarity determining region 3 (CDR3) is achieved using primers tohighly conserved sequences within the V and J regions of the gene locus,which may be used to identify clonal populations of cells for purposesof assessing minimal residual disease. Other methods for detectingclonal populations, such as single cell sequencing approaches, includingthose providing information regarding number of cells of a particularlineage and/or expressing a particular variable chain such as variableheavy chain or binding site thereof, such as a clonal population, may beused. In some aspects, the IGH DNA is amplified using a degenerateprimers or primers recognizing regions of variable chains shared amongdifferent cell clones, such as those recognizing consensus V anddegenerate consensus J region of the IGH sequence. An exemplary sequenceof the V region is ACACGGCCTCGTGTATTACTGT (SEQ ID NO: 57). An exemplarydegenerate consensus sequence of the J region is ACCTGAGGAGACGGTGACC(SEQ ID NO: 58).

The PCR product or sequencing result in some aspects is specific to therearranged allele and serves as a clonal marker for MRD detection.Following PCR amplification of the CDR3 region, PCR products can besequenced to yield patient-specific oligonucleotides constructed asprobes for allele-specific PCR for sensitive detection of MRD followingtreatment of B-cell malignancies with CAR-T cell therapy, e.g. CD19CAR-T cell therapy. In examples where a PCR product is not generatedusing the consensus primers, V region family-specific primers for theframework region 1 can be used instead.

In some aspects, persistence of PCR-detectable tumor cells such as cellsof the B cell malignancy such as the NHL or CLL, such as detectable IGHsequences corresponding to the malignant or clonal IGH sequences, aftertreatment is associated with increased risk of relapse. In some aspects,patients who are negative for malignant IGH sequences followingtreatment (in some aspects, even in the context of other criteriaindicating progressive disease or only a partial response, such aspersistence of enlarged lymph nodes or other criteria that may in somecontexts be associated with disease or lack of complete response) may bedeemed to have increased likelihood of PFS or to enter into CR ordurable CR or prolonged survival, compared to patients with persistentmalignant IGH sequences. In some embodiments, such prognostic andstaging determinations are particularly relevant for treatments in whichclearance of malignant cells is observed within a short period of timefollowing administration of the therapy, e.g., in comparison toresolution of other clinical symptoms such as lymph node size or otherstaging criteria. For example, in some such aspects, absence ofdetectable IGH or minimal residual disease in a sample such as the bonemarrow may be a preferred readout for response or likelihood of responseor durability thereof, as compared to other available staging orprognostic approaches. In some aspects, results from MRD, e.g., IGH deepsequencing information, may inform further intervention or lack thereof.For example, the methods and other provided embodiments in some contextsprovide that a subject deemed negative for malignant IGH may in someaspects be not further treated or not be further administered a dose ofthe therapy provided, or that the subject be administered a lower orreduced dose. Conversely, it may be provided or specified that a subjectexhibiting MRD via IGH deep sequencing be further treated, e.g., withthe therapy initially administered at a similar or higher dose or with afurther treatment. In some aspects, the disease or condition persistsfollowing administration of the first dose and/or administration of thefirst dose is not sufficient to eradicate the disease or condition inthe subject.

In some embodiments, the method reduces the burden of the disease orcondition, e.g., number of tumor cells, size of tumor, duration ofpatient survival or event-free survival, to a greater degree and/or fora greater period of time as compared to the reduction that would beobserved with a comparable method using an alternative dosing regimen,such as one in which the subject receives one or more alternativetherapeutic agents and/or one in which the subject does not receive adose of cells and/or a lymphodepleting agent in accord with the providedmethods, and/or with the provided articles of manufacture orcompositions. In some embodiments, the burden of a disease or conditionin the subject is detected, assessed, or measured. Disease burden may bedetected in some aspects by detecting the total number of disease ordisease-associated cells, e.g., tumor cells, in the subject, or in anorgan, tissue, or bodily fluid of the subject, such as blood or serum.In some aspects, survival of the subject, survival within a certain timeperiod, extent of survival, presence or duration of event-free orsymptom-free survival, or relapse-free survival, is assessed. In someembodiments, any symptom of the disease or condition is assessed. Insome embodiments, the measure of disease or condition burden isspecified.

In some embodiments, the event-free survival rate or overall survivalrate of the subject is improved by the methods, as compared with othermethods, for example, methods in which the subject receives one or morealternative therapeutic agents and/or one in which the subject does notreceive a dose of cells and/or a lymphodepleting agent in accord withthe provided methods, and/or with the provided articles of manufactureor compositions. For example, in some embodiments, event-free survivalrate or probability for subjects treated by the methods at 6 monthsfollowing the dose is greater than about 40%, greater than about 50%,greater than about 60%, greater than about 70%, greater than about 80%,greater than about 90%, or greater than about 95%. In some aspects,overall survival rate is greater than about 40%, greater than about 50%,greater than about 60%, greater than about 70%, greater than about 80%,greater than about 90%, or greater than about 95%. In some embodiments,the subject treated with the methods exhibits event-free survival,relapse-free survival, or survival to at least 6 months, or at least 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, the time toprogression is improved, such as a time to progression of greater thanat or about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10years.

In some embodiments, following treatment by the method, the probabilityof relapse is reduced as compared to other methods, for example, methodsin which the subject receives one or more alternative therapeutic agentsand/or one in which the subject does not receive a dose of cells and/ora lymphodepleting agent in accord with the provided methods, and/or withthe provided articles of manufacture or compositions. For example, insome embodiments, the probability of relapse at 6 months following thefirst dose is less than about 80%, less than about 70%, less than about60%, less than about 50%, less than about 40%, less than about 30%, lessthan about 20%, or less than about 10%.

In some cases, the pharmacokinetics of administered cells, e.g.,adoptively transferred cells are determined to assess the availability,e.g., bioavailability of the administered cells. Methods for determiningthe pharmacokinetics of adoptively transferred cells may include drawingperipheral blood from subjects that have been administered engineeredcells, and determining the number or ratio of the engineered cells inthe peripheral blood. Approaches for selecting and/or isolating cellsmay include use of chimeric antigen receptor (CAR)-specific antibodies(e.g., Brentjens et al., Sci. Transl. Med. 2013 March; 5(177): 177ra38)Protein L (Zheng et al., J. Transl. Med. 2012 February; 10:29), epitopetags, such as Strep-Tag sequences, introduced directly into specificsites in the CAR, whereby binding reagents for Strep-Tag are used todirectly assess the CAR (Liu et al. (2016) Nature Biotechnology, 34:430;international patent application Pub. No. WO2015095895) and monoclonalantibodies that specifically bind to a CAR polypeptide (seeinternational patent application Pub. No. WO2014190273). Extrinsicmarker genes may in some cases be utilized in connection with engineeredcell therapies to permit detection or selection of cells and, in somecases, also to promote cell suicide. A truncated epidermal growth factorreceptor (EGFRt) in some cases can be co-expressed with a transgene ofinterest (a CAR or TCR) in transduced cells (see e.g. U.S. Pat. No.8,802,374). EGFRt may contain an epitope recognized by the antibodycetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or bindingmolecule, which can be used to identify or select cells that have beenengineered with the EGFRt construct and another recombinant receptor,such as a chimeric antigen receptor (CAR), and/or to eliminate orseparate cells expressing the receptor. See U.S. Pat. No. 8,802,374 andLiu et al., Nature Biotech. 2016 April; 34(4): 430-434).

In some embodiments, the number of CAR⁺ T cells in a biological sampleobtained from the patient, e.g., blood, can be determined at a period oftime after administration of the cell therapy, e.g., to determine thepharmacokinetics of the cells. In some embodiments, number of CAR⁺ Tcells, optionally CAR⁺ CD8⁺ T cells and/or CAR⁺ CD4⁺ T cells, detectablein the blood of the subject, or in a majority of subjects so treated bythe method, is greater than 1 cells per μL, greater than 5 cells per μLor greater than per 10 cells per μL.

D. Toxicity

In some embodiments, the provided methods are designed to or includefeatures that result in a lower rate and/or lower degree of toxicity,toxic outcome or symptom, toxicity-promoting profile, factor, orproperty, such as a symptom or outcome associated with or indicative ofcytokine release syndrome (CRS) or neurotoxicity, for example, comparedto administration of an alternative cell therapy, such as an alternativeCAR⁺ T cell composition and/or an alternative dosing of cells, e.g. adosing of cells that is not administered at a defined ratio.

In some embodiments, the provided methods do not result in a high rateor likelihood of toxicity or toxic outcomes, or reduces the rate orlikelihood of toxicity or toxic outcomes, such as neurotoxicity (NT),cytokine release syndrome (CRS), such as compared to certain other celltherapies. In some embodiments, the methods do not result in, or do notincrease the risk of, severe NT (sNT), severe CRS (sCRS), macrophageactivation syndrome, tumor lysis syndrome, fever of at least at or about38 degrees Celsius for three or more days and a plasma level of CRP ofat least at or about 20 mg/dL. In some embodiments, greater than orgreater than about 30%, 35%, 40%, 50%, 55%, 60% or more of the subjectstreated according to the provided methods do not exhibit any grade ofCRS or any grade of neurotoxcity. In some embodiments, no more than 50%of subjects treated (e.g. at least 60%, at least 70%, at least 80%, atleast 90% or more of the subjects treated) exhibit a cytokine releasesyndrome (CRS) higher than grade 2 and/or a neurotoxicity higher thangrade 2. In some embodiments, at least 50% of subjects treated accordingto the method (e.g. at least 60%, at least 70%, at least 80%, at least90% or more of the subjects treated) do not exhibit a severe toxicoutcome (e.g. severe CRS or severe neurotoxicity), such as do notexhibit grade 3 or higher neurotoxicity and/or does not exhibit severeCRS, or does not do so within a certain period of time following thetreatment, such as within a week, two weeks, or one month of theadministration of the cells. In some embodiments, parameters assessed todetermine certain toxicities include adverse events (AEs), dose-limitingtoxicities (DLTs), CRS and NT.

Administration of adoptive T cell therapy, such as treatment with Tcells expressing chimeric antigen receptors, can induce toxic effects oroutcomes such as cytokine release syndrome and neurotoxicity. In someexamples, such effects or outcomes parallel high levels of circulatingcytokines, which may underlie the observed toxicity.

In some aspects, the toxic outcome is or is associated with orindicative of cytokine release syndrome (CRS) or severe CRS (sCRS). CRS,e.g., sCRS, can occur in some cases following adoptive T cell therapyand administration to subjects of other biological products. See Davilaet al., Sci Transl Med 6, 224ra25 (2014); Brentjens et al., Sci. Transl.Med. 5, 177ra38 (2013); Grupp et al., N. Engl. J. Med. 368, 1509-1518(2013); and Kochenderfer et al., Blood 119, 2709-2720 (2012); Xu et al.,Cancer Letters 343 (2014) 172-78.

Typically, CRS is caused by an exaggerated systemic immune responsemediated by, for example, T cells, B cells, NK cells, monocytes, and/ormacrophages. Such cells may release a large amount of inflammatorymediators such as cytokines and chemokines. Cytokines may trigger anacute inflammatory response and/or induce endothelial organ damage,which may result in microvascular leakage, heart failure, or death.Severe, life-threatening CRS can lead to pulmonary infiltration and lunginjury, renal failure, or disseminated intravascular coagulation. Othersevere, life-threatening toxicities can include cardiac toxicity,respiratory distress, neurologic toxicity and/or hepatic failure.

CRS may be treated using anti-inflammatory therapy such as an anti-IL-6therapy, e.g., anti-IL-6 antibody, e.g., tocilizumab, or antibiotics orother agents as described. Outcomes, signs and symptoms of CRS are knownand include those described herein. In some embodiments, where aparticular dosage regimen or administration effects or does not effect agiven CRS-associated outcome, sign, or symptom, particular outcomes,signs, and symptoms and/or quantities or degrees thereof may bespecified.

In the context of administering CAR-expressing cells, CRS typicallyoccurs 6-20 days after infusion of cells that express a CAR. See Xu etal., Cancer Letters 343 (2014) 172-78. In some cases, CRS occurs lessthan 6 days or more than 20 days after CAR T cell infusion. Theincidence and timing of CRS may be related to baseline cytokine levelsor tumor burden at the time of infusion. Commonly, CRS involves elevatedserum levels of interferon (IFN)-γ, tumor necrosis factor (TNF)-α,and/or interleukin (IL)-2. Other cytokines that may be rapidly inducedin CRS are IL-1β, IL-6, IL-8, and IL-10.

Exemplary outcomes associated with CRS include fever, rigors, chills,hypotension, dyspnea, acute respiratory distress syndrome (ARDS),encephalopathy, ALT/AST elevation, renal failure, cardiac disorders,hypoxia, neurologic disturbances, and death. Neurological complicationsinclude delirium, seizure-like activity, confusion, word-findingdifficulty, aphasia, and/or becoming obtunded. Other CRS-relatedoutcomes include fatigue, nausea, headache, seizure, tachycardia,myalgias, rash, acute vascular leak syndrome, liver function impairment,and renal failure. In some aspects, CRS is associated with an increasein one or more factors such as serum-ferritin, d-dimer,aminotransferases, lactate dehydrogenase and triglycerides, or withhypofibrinogenemia or hepatosplenomegaly. Other exemplary signs orsymptoms associated with CRS include hemodynamic instability, febrileneutropenia, increase in serum C-reactive protein (CRP), changes incoagulation parameters (for example, international normalized ratio(INR), prothrombin time (PTI) and/or fibrinogen), changes in cardiac andother organ function, and/or absolute neutrophil count (ANC).

In some embodiments, outcomes associated with CRS include one or moreof: persistent fever, e.g., fever of a specified temperature, e.g.,greater than at or about 38 degrees Celsius, for two or more, e.g.,three or more, e.g., four or more days or for at least three consecutivedays; fever greater than at or about 38 degrees Celsius; elevation ofcytokines, such as a max fold change, e.g., of at least at or about 75,compared to pre-treatment levels of at least two cytokines (e.g., atleast two of the group consisting of interferon gamma (IFNγ), GM-CSF,IL-6, IL-10, Flt-3L, fracktalkine, and IL-5, and/or tumor necrosisfactor alpha (TNFα)), or a max fold change, e.g., of at least at orabout 250 of at least one of such cytokines; and/or at least oneclinical sign of toxicity, such as hypotension (e.g., as measured by atleast one intravenous vasoactive pressor); hypoxia (e.g., plasma oxygen(PO₂) levels of less than at or about 90%); and/or one or moreneurologic disorders (including mental status changes, obtundation, andseizures).

Exemplary CRS-related outcomes include increased or high serum levels ofone or more factors, including cytokines and chemokines and otherfactors associated with CRS. Exemplary outcomes further includeincreases in synthesis or secretion of one or more of such factors. Suchsynthesis or secretion can be by the T cell or a cell that interactswith the T cell, such as an innate immune cell or B cell.

In some embodiments, the CRS-associated serum factors or CRS-relatedoutcomes include inflammatory cytokines and/or chemokines, includinginterferon gamma (IFN-γ), TNF-a, IL-1β, IL-2, IL-6, IL-7, IL-8, IL-10,IL-12, sIL-2Ra, granulocyte macrophage colony stimulating factor(GM-CSF), macrophage inflammatory protein (MIP)-1, tumor necrosis factoralpha (TNFα), IL-6, and IL-10, IL-1β, IL-8, IL-2, MIP-1, Flt-3L,fracktalkine, and/or IL-5. In some embodiments, the factor or outcomeincludes C reactive protein (CRP). In addition to being an early andeasily measurable risk factor for CRS, CRP also is a marker for cellexpansion. In some embodiments, subjects that are measured to have highlevels of CRP, such as ≥15 mg/dL, have CRS. In some embodiments,subjects that are measured to have high levels of CRP do not have CRS.In some embodiments, a measure of CRS includes a measure of CRP andanother factor indicative of CRS.

In some embodiments, one or more inflammatory cytokines or chemokinesare monitored before, during, or after CAR treatment. In some aspects,the one or more cytokines or chemokines include IFN-γ, TNF-α, IL-2,IL-1β, IL-6, IL-7, IL-8, IL-10, IL-12, sIL-2Rα, granulocyte macrophagecolony stimulating factor (GM-CSF), or macrophage inflammatory protein(MIP). In some embodiments, IFN-γ, TNF-α, and IL-6 are monitored.

CRS criteria that appear to correlate with the onset of CRS to predictwhich patients are more likely to be at risk for developing sCRS havebeen developed (see Davilla et al. Science translational medicine. 2014;6(224):224ra25). Factors include fevers, hypoxia, hypotension,neurologic changes, elevated serum levels of inflammatory cytokines,such as a set of seven cytokines (IFNγ, IL-5, IL-6, IL-10, Flt-3L,fractalkine, and GM-CSF) whose treatment-induced elevation can correlatewell with both pretreatment tumor burden and sCRS symptoms. Otherguidelines on the diagnosis and management of CRS are known (see e.g.,Lee et al, Blood. 2014; 124(2):188-95). In some embodiments, thecriteria reflective of CRS grade are those detailed in Table 3 below.

TABLE 3 Exemplary Grading Criteria for CRS Grade Description of Symptoms1 Not life-threatening, require only symptomatic treatment such asantipyretics Mild and anti-emetics (e.g., fever, nausea, fatigue,headache, myalgias, malaise) 2 Require and respond to moderateintervention: Moderate Oxygen requirement <40%, or Hypotensionresponsive to fluids or low dose of a single vasopressor, or Grade 2organ toxicity (by CTCAE v4.0) 3 Require and respond to aggressiveintervention: Severe Oxygen requirement ≥40%, or Hypotension requiringhigh dose of a single vasopressor (e.g., norepinephrine ≥20 μg/kg/min,dopamine ≥10 μg/kg/min, phenylephrine ≥200 μg/kg/min, or epinephrine ≥10μg/kg/min), or Hypotension requiring multiple vasopressors (e.g.,vasopressin + one of the above agents, or combination vasopressorsequivalent to ≥20 μg/kg/min norepinephrine), or Grade 3 organ toxicityor Grade 4 transaminitis (by CTCAE v4.0) 4 Life-threatening:Life-threatening Requirement for ventilator support, or Grade 4 organtoxicity (excluding transaminitis) 5 Death Fatal

In some embodiments, a subject is deemed to develop “severe CRS”(“sCRS”) in response to or secondary to administration of a cell therapyor dose of cells thereof, if, following administration, the subjectdisplays: (1) fever of at least 38 degrees Celsius for at least threedays; (2) cytokine elevation that includes either (a) a max fold changeof at least 75 for at least two of the following group of sevencytokines compared to the level immediately following theadministration: interferon gamma (IFNγ), GM-CSF, IL-6, IL-10, Flt-3L,fracktalkine, and IL-5 and/or (b) a max fold change of at least 250 forat least one of the following group of seven cytokines compared to thelevel immediately following the administration: interferon gamma (IFNγ),GM-CSF, IL-6, IL-10, Flt-3L, fracktalkine, and IL-5; and (c) at leastone clinical sign of toxicity such as hypotension (requiring at leastone intravenous vasoactive pressor) or hypoxia (PO₂<90%) or one or moreneurologic disorder(s) (including mental status changes, obtundation,and/or seizures). In some embodiments, severe CRS includes CRS with agrade of 3 or greater, such as set forth in Table 3.

In some embodiments, outcomes associated with severe CRS or grade 3 CRSor greater, such as grade 4 or greater, include one or more of:persistent fever, e.g., fever of a specified temperature, e.g., greaterthan at or about 38 degrees Celsius, for two or more, e.g., three ormore, e.g., four or more days or for at least three consecutive days;fever greater than at or about 38 degrees Celsius; elevation ofcytokines, such as a max fold change, e.g., of at least at or about 75,compared to pre-treatment levels of at least two cytokines (e.g., atleast two of the group consisting of interferon gamma (IFNγ), GM-CSF,IL-6, IL-10, Flt-3L, fracktalkine, and IL-5, and/or tumor necrosisfactor alpha (TNFα)), or a max fold change, e.g., of at least at orabout 250 of at least one of such cytokines; and/or at least oneclinical sign of toxicity, such as hypotension (e.g., as measured by atleast one intravenous vasoactive pressor); hypoxia (e.g., plasma oxygen(PO₂) levels of less than at or about 90%); and/or one or moreneurologic disorders (including mental status changes, obtundation, andseizures). In some embodiments, severe CRS includes CRS that requiresmanagement or care in the intensive care unit (ICU).

In some embodiments, the CRS, such as severe CRS, encompasses acombination of (1) persistent fever (fever of at least 38 degreesCelsius for at least three days) and (2) a serum level of CRP of atleast at or about 20 mg/dL. In some embodiments, the CRS encompasseshypotension requiring the use of two or more vasopressors or respiratoryfailure requiring mechanical ventilation. In some embodiments, thedosage of vasopressors is increased in a second or subsequentadministration.

In some embodiments, severe CRS or grade 3 CRS encompasses an increasein alanine aminotransferase, an increase in aspartate aminotransferase,chills, febrile neutropenia, headache, left ventricular dysfunction,encephalopathy, hydrocephalus, and/or tremor.

The method of measuring or detecting the various outcomes may bespecified.

In some aspects, the toxic outcome is or is associated withneurotoxicity. In some embodiments, symptoms associated with a clinicalrisk of neurotoxicity include confusion, delirium, aphasia, expressiveaphasia, obtundation, myoclonus, lethargy, altered mental status,convulsions, seizure-like activity, seizures (optionally as confirmed byelectroencephalogram [EEG]), elevated levels of beta amyloid (Aβ),elevated levels of glutamate, and elevated levels of oxygen radicals. Insome embodiments, neurotoxicity is graded based on severity (e.g., usinga Grade 1-5 scale (see, e.g., Guido Cavaletti & Paola Marmiroli NatureReviews Neurology 6, 657-666 (December 2010); National CancerInstitute-Common Toxicity Criteria version 4.03 (NCI-CTCAE v4.03).

In some instances, neurologic symptoms may be the earliest symptoms ofsCRS. In some embodiments, neurologic symptoms are seen to begin 5 to 7days after cell therapy infusion. In some embodiments, duration ofneurologic changes may range from 3 to 19 days. In some cases, recoveryof neurologic changes occurs after other symptoms of sCRS have resolved.In some embodiments, time or degree of resolution of neurologic changesis not hastened by treatment with anti-IL-6 and/or steroid(s).

In some embodiments, a subject is deemed to develop “severeneurotoxicity” in response to or secondary to administration of a celltherapy or dose of cells thereof, if, following administration, thesubject displays symptoms that limit self-care (e.g. bathing, dressingand undressing, feeding, using the toilet, taking medications) fromamong: 1) symptoms of peripheral motor neuropathy, includinginflammation or degeneration of the peripheral motor nerves; 2) symptomsof peripheral sensory neuropathy, including inflammation or degenerationof the peripheral sensory nerves, dysesthesia, such as distortion ofsensory perception, resulting in an abnormal and unpleasant sensation,neuralgia, such as intense painful sensation along a nerve or a group ofnerves, and/or paresthesia, such as functional disturbances of sensoryneurons resulting in abnormal cutaneous sensations of tingling,numbness, pressure, cold and warmth in the absence of stimulus. In someembodiments, severe neurotoxicity includes neurotoxicity with a grade of3 or greater, such as set forth in Table 4.

TABLE 4 Exemplary Grading Criteria for neurotoxicity Grade Descriptionof Symptoms 1 Mild or asymptomatic symptoms Asymptomatic or Mild 2Presence of symptoms that limit instrumental Moderate activities ofdaily living (ADL), such as preparing meals, shopping for groceries orclothes, using the telephone, managing money 3 Presence of symptoms thatlimit self-care ADL, Severe such as bathing, dressing and undressing,feeding self, using the toilet, taking medications 4 Symptoms that arelife-threatening, requiring Life-threatening urgent intervention 5 DeathFatal

In some embodiments, the methods reduce symptoms associated with CRS orneurotoxicity compared to other methods. In some aspects, the providedmethods reduce symptoms, outcomes or factors associated with CRS,including symptoms, outcomes or factors associated with severe CRS orgrade 3 or higher CRS, compared to other methods. For example, subjectstreated according to the present methods may lack detectable and/or havereduced symptoms, outcomes or factors of CRS, e.g. severe CRS or grade 3or higher CRS, such as any described, e.g. set forth in Table 3. In someembodiments, subjects treated according to the present methods may havereduced symptoms of neurotoxicity, such as limb weakness or numbness,loss of memory, vision, and/or intellect, uncontrollable obsessiveand/or compulsive behaviors, delusions, headache, cognitive andbehavioral problems including loss of motor control, cognitivedeterioration, and autonomic nervous system dysfunction, and sexualdysfunction, compared to subjects treated by other methods. In someembodiments, subjects treated according to the present methods may havereduced symptoms associated with peripheral motor neuropathy, peripheralsensory neuropathy, dysethesia, neuralgia or paresthesia.

In some embodiments, the methods reduce outcomes associated withneurotoxicity including damages to the nervous system and/or brain, suchas the death of neurons. In some aspects, the methods reduce the levelof factors associated with neurotoxicity such as beta amyloid (Aβ),glutamate, and oxygen radicals.

In some embodiments, the toxicity outcome is a dose-limiting toxicity(DLT). In some embodiments, the toxic outcome is a dose-limitingtoxicity. In some embodiments, the toxic outcome is the absence of adose-limiting toxicity. In some embodiments, a dose-limiting toxicity(DLT) is defined as any grade 3 or higher toxicity as assessed by anyknown or published guidelines for assessing the particular toxicity,such as any described above and including the National Cancer Institute(NCI) Common Terminology Criteria for Adverse Events (CTCAE) version4.0.

In some embodiments, the low rate, risk or likelihood of developing atoxicity, e.g. CRS or neurotoxicity or severe CRS or neurotoxicity, e.g.grade 3 or higher CRS or neurotoxicity, observed with administering adose of T cells in accord with the provided methods, and/or with theprovided articles of manufacture or compositions, permits administrationof the cell therapy on an outpatient basis. In some embodiments, theadministration of the cell therapy, e.g. dose of T cells (e.g. CAR⁺ Tcells) in accord with the provided methods, and/or with the providedarticles of manufacture or compositions, is performed on an outpatientbasis or does not require admission to the subject to the hospital, suchas admission to the hospital requiring an overnight stay.

In some aspects, subjects administered the cell therapy, e.g. dose of Tcells (e.g. CAR⁺ T cells) in accord with the provided methods, and/orwith the provided articles of manufacture or compositions, includingsubjects treated on an outpatient basis, are not administered anintervention for treating any toxicity prior to or with administrationof the cell dose, unless or until the subject exhibits a sign or symptomof a toxicity, such as of a neurotoxicity or CRS. Exemplary agents fortreating, delaying, attenuating or ameliorating a toxicity are describedin Section II.

In some embodiments, if a subject administered the cell therapy, e.g.dose of T cells (e.g. CAR⁺ T cells), including subjects treated on anoutpatient basis, exhibits a fever the subject is given or is instructedto receive or administer a treatment to reduce the fever. In someembodiments, the fever in the subject is characterized as a bodytemperature of the subject that is (or is measured at) at or above acertain threshold temperature or level. In some aspects, the thresholdtemperature is that associated with at least a low-grade fever, with atleast a moderate fever, and/or with at least a high-grade fever. In someembodiments, the threshold temperature is a particular temperature orrange. For example, the threshold temperature may be at or about or atleast at or about 38, 39, 40, 41, or 42 degrees Celsius, and/or may be arange of at or about 38 degrees Celsius to at or about 39 degreesCelsius, a range of at or about 39 degrees Celsius to at or about 40degrees Celsius, a range of at or about 40 degrees Celsius to at orabout 41 degrees, or a range of at or about 41 degrees Celsius to at orabout 42 degrees Celsius.

In some embodiments, the treatment designed to reduce fever includestreatment with an antipyretic. An antipyretic may include any agent,e.g., compound, composition, or ingredient, that reduces fever, such asone of any number of agents known to have antipyretic effects, such asNSAIDs (such as ibuprofen, naproxen, ketoprofen, and nimesulide),salicylates, such as aspirin, choline salicylate, magnesium salicylate,and sodium salicylate, paracetamol, acetaminophen, Metamizole,Nabumetone, Phenaxone, antipyrine, febrifuges. In some embodiments, theantipyretic is acetaminophen. In some embodiments, acetaminophen can beadministered at a dose of 12.5 mg/kg orally or intravenously up to everyfour hours. In some embodiments, it is or comprises ibuprofen oraspirin.

In some embodiments, if the fever is a sustained fever, the subject isadministered an alternative treatment for treating the toxicity, such asany described in Section II below. For subjects treated on an outpatientbasis, the subject is instructed to return to the hospital if thesubject has and/or is determined to or to have a sustained fever. Insome embodiments, the subject has, and/or is determined to or consideredto have, a sustained fever if he or she exhibits a fever at or above therelevant threshold temperature, and where the fever or body temperatureof the subject is not reduced, or is not reduced by or by more than aspecified amount (e.g., by more than 1° C., and generally does notfluctuate by about, or by more than about, 0.5° C., 0.4° C., 0.3° C., or0.2° C.), following a specified treatment, such as a treatment designedto reduce fever such as treatment with an antipyreticm, e.g. NSAID orsalicylates, e.g. ibuprofen, acetaminophen or aspirin. For example, asubject is considered to have a sustained fever if he or she exhibits oris determined to exhibit a fever of at least at or about 38 or 39degrees Celsius, which is not reduced by or is not reduced by more thanat or about 0.5° C., 0.4° C., 0.3° C., or 0.2° C., or by at or about 1%,2%, 3%, 4%, or 5%, over a period of 6 hours, over a period of 8 hours,or over a period of 12 hours, or over a period of 24 hours, evenfollowing treatment with the antipyretic such as acetaminophen. In someembodiments, the dosage of the antipyretic is a dosage ordinarilyeffective in such as subject to reduce fever or fever of a particulartype such as fever associated with a bacterial or viral infection, e.g.,a localized or systemic infection.

In some embodiments, the subject has, and/or is determined to orconsidered to have, a sustained fever if he or she exhibits a fever ator above the relevant threshold temperature, and where the fever or bodytemperature of the subject does not fluctuate by about, or by more thanabout, 1° C., and generally does not fluctuate by about, or by more thanabout, 0.5° C., 0.4° C., 0.3° C., or 0.2° C. Such absence of fluctuationabove or at a certain amount generally is measured over a given periodof time (such as over a 24-hour, 12-hour, 8-hour, 6-hour, 3-hour, or1-hour period of time, which may be measured from the first sign offever or the first temperature above the indicated threshold). Forexample, in some embodiments, a subject is considered to or isdetermined to exhibit sustained fever if he or she exhibits a fever ofat least at or about or at least at or about 38 or 39 degrees Celsius,which does not fluctuate in temperature by more than at or about 0.5°C., 0.4° C., 0.3° C., or 0.2° C., over a period of 6 hours, over aperiod of 8 hours, or over a period of 12 hours, or over a period of 24hours.

In some embodiments, the fever is a sustained fever; in some aspects,the subject is treated at a time at which a subject has been determinedto have a sustained fever, such as within one, two, three, four, fivesix, or fewer hours of such determination or of the first suchdetermination following the initial therapy having the potential toinduce the toxicity, such as the cell therapy, such as dose of T cells,e.g. CAR⁺ T cells.

In some embodiments, one or more interventions or agents for treatingthe toxicity, such as a toxicity-targeting therapies, is administered ata time at which or immediately after which the subject is determined toor confirmed to (such as is first determined or confirmed to) exhibitsustained fever, for example, as measured according to any of theaforementioned embodiments. In some embodiments, the one or moretoxicity-targeting therapies is administered within a certain period oftime of such confirmation or determination, such as within 30 minutes, 1hour, 2 hours, 3 hours, 4 hours, 6 hours, or 8 hours thereof.

E. Biomarkers, Analytes or Parameters

Among the provided methods are methods of assessing a risk fordeveloping toxicity associated with cell therapy in a subject thatinvolves assessing or detecting biomarkers (e.g., analytes) orparameters that are associated with the toxicity, e.g., neurotoxicity,such as severe neurotoxicity, and/or CRS, such as severe CRS. Also amongthe provided methods are methods of assessing the likelihood of responseto a cell therapy in a subject that involves assessing or detectingbiomarkers (e.g., analytes) or parameters that are associated with aresponse outcome, such as objective response (OR), including completeresponse (CR) and partial response (PR). In some embodiments, theassociate response outcome includes durable response, such as a responsethat is durable for 3 months, 6 months, 9 months 12 months or more,after the initial response.

In some embodiments, the methods involve assessing or detecting thepresence or absence of one or a panel of biomarkers (e.g. analytes)and/or parameters (e.g. concentration, amount, level or activity)associated with one or a panel of biomarkers (e.g. analytes). In somecases, the methods can include comparing the one or more parameters to aparticular reference value, such as a threshold level (also called“threshold value” herein), e.g., those associated with a risk fordeveloping toxicity or those associated with a particular response, suchas OR, CR or PR, or durable response, such as a response that is durablefor 3 months, 6 months, 9 months 12 months or more, after the initialresponse. In some embodiments, the methods also involve selectingsubjects for treatment with a cell therapy based on the assessment ofthe presence or absence of the biomarker and/or comparison of thebiomarkers to a reference value or threshold level of the biomarker. Insome embodiments, the methods also involve administering an agent or atherapy that can treat, prevent, delay and/or attenuate development ofthe toxicity, e.g., based on the assessment of the presence or absenceof the biomarker and/or comparison of the biomarkers to a referencevalue or threshold level of the biomarker.

In some embodiments, the methods involve assessing the likelihood ofresponse of the subject or the risk of development of a toxicity, afteradministration of a cell therapy. In some embodiments, the methodsinvolve assessing the level, amount or concentration of one or moreanalyte in a biological sample, wherein the biological sample is from asubject that is a candidate for treatment with the cell therapy, saidcell therapy optionally comprising a dose or composition of geneticallyengineered cells expressing a recombinant receptor; and the biologicalsample is obtained from the subject prior to administering the celltherapy and/or said biological sample does not comprise the recombinantreceptor and/or said engineered cells. In some aspects, the methodsinvolve comparing, individually, the level, amount or concentration ofthe analyte in the sample to a threshold level, thereby determining arisk of developing a toxicity after administration of the cell therapy.In some aspects, the comparisons can be used to determine the likelihoodof response of the subject or the risk of development of a toxicity,after administration of a cell therapy.

In some embodiments, the methods also involve selecting subjects fortreatment with an a cell therapy, such as a particular dose of celltherapy, including administration of a particular dose of cell therapysuch as those described herein, e.g., in Section I.A and I.B, based onthe assessment of the presence or absence of the biomarker and/orcomparison of the biomarkers to a reference value or threshold level ofthe biomarker. In some embodiments, the methods also involve selectingsubjects for treatment with an additional agent, such as an agent orother treatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity, basedon the assessment of the presence or absence of the biomarker and/orcomparison of the biomarkers to a reference value or threshold level ofthe biomarker.

In some embodiments, the parameter is or includes attributes, factors,characteristic of the patient and/or the disease or condition. In someembodiments, the parameter is a parameter related to tumor burden, e.g.,a measurement of tumor burden. In some aspects, the methods also involvefurther monitoring the subject for possible symptoms of toxicity basedon the risk of toxicity determined by assessment of the presence orabsence of the biomarker and/or comparison of the biomarkers to areference value or threshold level of the biomarker. In some aspects, abiological sample, e.g., blood sample or tissue sample from the subject,can be obtained for detecting the presence or absence of a biomarker(e.g. analyte), such as for detecting or measuring a parameter (e.g.concentration, amount, level or activity) of the biomarker and/orassessing the presence of a biomarker, for analysis, correlation and/ordetection of particular outcomes and/or toxicities. In some embodiments,certain physiological or biological parameters associated with abiomarker, including expression of biomarkers and/or clinical andlaboratory parameters, can be assessed, from a biological sample, e.g.,blood, from subjects before or after administration of the cell therapy.In some embodiments, expression biomarkers or analytes and/or clinicaland laboratory parameters, can be assessed from a biological sample,e.g., blood, from subjects before administration of the cell therapy(pre-treatment). In some embodiments, expression biomarkers or analytesand/or clinical and laboratory parameters, can be assessed from abiological sample, e.g., blood, from subjects after administration ofthe cell therapy (post-treatment). In some embodiments, theconcentration, amount, level or activity of biomarkers (e.g., analytes)and/or clinical and laboratory parameters can be assessed at one or moretime points before or after administration of the cell therapy. In someembodiments, the peak concentration, amount, level or activity ofbiomarkers (e.g., analytes) and/or clinical and laboratory parametersduring a specified period of time can also be determined.

In some embodiments, a biomarker or an analyte is an objectivelymeasurable characteristic or a molecule expressed by or in a biologicalsample, including cells, that can be indicative of or associated with aparticular state or phenomenon, such as a biological process, atherapeutic outcome; a cell phenotype or a diseased state. In someaspects, a biomarker or an analyte or parameters associated with abiomarker or an analyte can be measured or detected. For example, thepresence or absence of expression of a biomarker or analyte, can bedetected. In some aspects, the parameters such as concentration, amount,level or activity of the biomarker or analyte can be measured ordetected. In some embodiments, the presence, absence, expression,concentration, amount, level and/or activity of the biomarker can beassociated with, correlated to, indicative of and/or predictive ofparticular states, such as particular therapeutic outcomes or state ofthe subject. In some aspects, the presence, absence, expression,concentration, amount, level and/or activity of the biomarker oranalyte, such as any described herein, can be used to assess thelikelihood of a particular outcome or state, such as a particulartherapeutic outcome, including response outcome or toxicity outcome. Insome embodiments, exemplary biomarkers include cytokines, cell surfacemolecules, chemokines, receptors, soluble receptors, soluble serumproteins and/or degradation products. In some embodiments, biomarkers oranalytes can also include particular attributes, factors, characteristicof the patient and/or the disease or condition or factors indicative ofthe state of the patient and/or the disease or condition of the patient(including disease burden), and/or clinical or laboratory parameters.

In some embodiments, the biomarkers can be used singly or in combinationwith other biomarkers, such as in a panel of biomarkers. In someembodiments, expression of particular biomarkers can be correlated toparticular outcomes or toxicities, e.g., development of neurotoxicity.In some embodiments, biomarkers (e.g. analytes), including parametersthereof, that can be assessed include Lactate dehydrogenase (LDH),ferritin, C-reactive protein (CRP), Interleukin-6 (IL-6), IL-7, IL-8,IL-10, IL-15, IL-16, tumor necrosis factor alpha (TNF-α), interferonalpha 2 (IFN-α2), monocyte chemoattractant protein-1 (MCP-1), macrophageinflammatory protein 1 alpha (MIP-1α), macrophage inflammatory protein 1beta (MIP-1β), Eotaxin, Granulocyte-colony stimulating factor (G-CSF),IL-1 receptor alpha (IL-1Rα), IL-1β, IFN-γ-Inducible Protein 10 (IP-10),perforin, and D-dimer (fibrin degradation product). In some embodiments,the biomarkers (e.g. analytes), including parameters thereof, includeLDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 and MIP-1β.In some embodiments, the biomarkers (e.g. analytes), includingparameters thereof, include ferritin, CRP, D-dimer, IL-6, IL-15, TNF-αand MIP-1α. In some embodiments, the biomarkers (e.g. analytes),including parameters thereof, include ferritin, CRP, IL-10, IL-15,IL-16, TNF-α, or MIP-1β.

In some embodiments, the methods include detecting the presence orabsence of one or more biomarkers, such as a parameter (e.g.concentration, amount, level or activity) associated with one or morebiomarkers, in which the one or more biomarkers are selected from amongLactate dehydrogenase (LDH), ferritin, C-reactive protein (CRP),Interleukin-6 (IL-6), IL-7, IL-8, IL-10, IL-15, IL-16, tumor necrosisfactor alpha (TNF-α), interferon alpha 2 (IFN-α2), monocytechemoattractant protein-1 (MCP-1), macrophage inflammatory protein 1alpha (MIP-1α), macrophage inflammatory protein 1 beta (MIP-1β),Eotaxin, Granulocyte-colony stimulating factor (G-CSF), IL-1 receptoralpha (IL-1Rα), IL-10, IFN-γ-Inducible Protein 10 (IP-10), perforin, andD-dimer (fibrin degradation product).

In some embodiments, the parameter that is assessed is or includesattributes, factors, characteristic of the patient and/or the disease orcondition, and/or expression of biomarkers. In some embodiments, theparameter is or includes one or more factors indicative of the state ofthe patient and/or the disease or condition of the patient. In someembodiments, the parameter is indicative of tumor burden. In someembodiments, the factor indicative of tumor burden is a volumetricmeasure of tumor(s). In some embodiments, the volumetric measure is ameasure of the lesion(s), such as the tumor size, tumor diameter, tumorvolume, tumor mass, tumor load or bulk, tumor-related edema,tumor-related necrosis, and/or number or extent of metastases. In someembodiments, the volumetric measure of tumor is a bidimensional measure.For example, in some embodiments, the area of lesion(s) are calculatedas the product of the longest diameter and the longest perpendiculardiameter of all measurable tumors. In some cases, the volumetric measureof tumor is a unidimensional measure. In some cases, the size ofmeasurable lesions is assessed as the longest diameter. In someembodiments, the sum of the products of diameters (SPD), longest tumordiameters (LD), sum of longest tumor diameters (SLD), necrosis, tumorvolume, necrosis volume, necrosis-tumor ratio (NTR), peritumoral edema(PTE), and edema-tumor ratio (ETR) is measured.

Exemplary methods for measuring and assessing tumor burden include thosedescribed in, e.g., Carceller et al., Pediatr Blood Cancer. (2016)63(8):1400-1406 and Eisenhauer et al., Eur J Cancer. (2009)45(2):228-247. In some embodiments, the volumetric is a sum of theproducts of diameters (SPD) measured by determining the sum of theproducts of the largest perpendicular diameters of all measurabletumors. In some aspects, the tumor or lesion are measured in onedimension with the longest diameter (LD) and/or by determining the sumof longest tumor diameters (SLD) of all measurable lesions. In someembodiments, the volumetric measure of tumor is a volumetricquantification of tumor necrosis, such as necrosis volume and/ornecrosis-tumor ratio (NTR), see Monsky et al., Anticancer Res. (2012)32(11): 4951-4961. In some aspects, the volumetric measure of tumor is avolumetric quantification of tumor-related edema, such as peritumoraledema (PTE) and/or edema-tumor ratio (ETR). In some embodiments,measuring can be performed using imaging techniques such as computedtomography (CT), positron emission tomography (PET), and/or magneticresonance imaging (MRI) of the subject.

In some embodiments, the volumetric measure of tumor is determined at ascreening session, such as a routine assessment or blood draw to confirmand/or identify the condition or disease in the subject.

In some embodiments, the presence or absence and/or a parameter of oneor more biomarkers (e.g. analytes) is assessed from a biological sample.In some aspects, the biological sample is a bodily fluid or a tissue. Insome such embodiments, the biological sample, e.g., bodily fluid, is orcontains whole blood, serum or plasma.

In some embodiments, the presence or absence and/or a parameter of oneor more biomarkers (e.g. analytes) is assessed prior to administrationof the cell therapy (e.g., pre-infusion), e.g., obtained up to 2 days,up to 7 days, up to 14 days, up to 21 days, up to 28 days, up to 35 daysor up to 40 days prior to initiation of the administration of theengineered cells. In some embodiments, the biological sample is obtainedfrom the subject prior to administration of the cell therapy (e.g.,pre-infusion), e.g., obtained up to 2 days, up to 7 days, up to 14 days,up to 21 days, up to 28 days, up to 35 days or up to 40 days prior toinitiation of the administration of the engineered cells.

In some embodiments, the biological sample is an apheresis orleukaphresis sample. In some embodiments, the or absence and/or aparameter of one or more biomarkers (e.g. analytes) is assessed or thebiological sample is obtained after administration of the cell therapy.In some embodiments, the reagents can be used prior to theadministration of the cell therapy or after the administration of celltherapy, for diagnostic purposes, to identify subjects and/or to assesstreatment outcomes and/or toxicities.

In some embodiments, measuring the value of the one or more biomarkerscomprises performing an in vitro assay. In some aspects, the in vitroassay is an immunoassay, an aptamer-based assay, a histological orcytological assay, or an mRNA expression level assay. In someembodiments, the values of the one or more biomarkers are measured by anenzyme-linked immunosorbent assay (ELISA), immunoblotting,immunoprecipitation, radioimmunoassay (MA), immunostaining, a flowcytometry assay, surface plasmon resonance (SPR), a chemiluminescenceassay, a lateral flow immunoassay, an inhibition assay or an avidityassay. In some cases, the value of at least one of the one or morebiomarkers is determined using a binding reagent that specifically bindsto at least one biomarker. In some aspects, the binding reagent is anantibody or antigen-binding fragment thereof, an aptamer or a nucleicacid probe.

In some embodiments, measuring the value of the one or more biomarkers(e.g., analytes) comprises contacting a reagent capable of directly orindirectly detecting the analyte with the biological sample anddetermining the presence or absence, level, amount or concentration ofthe analyte in the biological sample. In some embodiments, the one ormore biomarker (e.g. analyte) is lactate dehydrogenase (LDH), ferritin,CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1,MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, andD-dimer (fibrin degradation product). In some embodiments, the one ormore biomarker (e.g. analyte) is LDH, ferritin, CRP, IL-6, IL-8, IL-10,TNF-alpha, IFN-alpha2, MCP-1 and MIP-1beta. In some embodiments, the oneor more biomarker (e.g. analyte) is or includes LDH.

In some aspects, the reagent is a binding molecule that specificallybinds to the analyte. For example, in some embodiments, the reagent isan antibody or an antigen-binding fragment thereof. In some embodiments,the reagent is or includes a substrate or binding partner of theanalyte.

In some embodiments, the presence, absence or parameter (e.g. level,amount, concentration and/or other measure) of LDH is detected ordetermined in a sample. Various methods of detecting or determining LDHare known. For example, an assay which measures LDH conversion oflactate to pyruvate through NAD⁺ reduction to NADH can be used to detectLDH in the sample. In some embodiments, the sample is contacted withlactate in the presence of coenzyme NAD which, as a measure of LDH inthe sample, results in NADH that is then oxidized in the presence of anelectron transfer agent. In some embodiments, the NADH interacts with aprobe or dye precursor that is detectable by measuring absorption in avisible light range. In some examples, diaphorase uses the NADH toreduce tetrazolium salt (INT) to a red formazan product and the productis measured. Therefore, in some embodiments, the amount of coloredproduct formed is directly proportional to the LDH activity in thesample.

In some embodiments, the methods involve comparing, individually, thelevel, amount or concentration of the analyte in the sample to athreshold level, thereby determining a risk of developing a toxicityafter administration of the cell therapy, or thereby determining alikelihood that a subject will achieve a response to the cell therapy.In some aspects, the exemplary threshold levels can be determined basedon the mean or median values and values within a range or standarddeviation of the mean or median values of the level, amount orconcentration of the analyte in a biological sample obtained from agroup of subjects prior to receiving a cell therapy, wherein each of thesubjects of the group went on to exhibit a particular outcome, such as aparticular therapeutic outcome, including either exhibiting a responseor not exhibiting a response; or either developing a toxicity or notdeveloping a toxicity. In some embodiments, particular aspects ofdetermining threshold values include those described below in SectionsI.E.1 and I.E.2.

1. Exemplary Biomarkers, Analytes or Parameters Associated with ResponseOutcomes

In some embodiments, the analyte or biomarker is associated with,correlated to, indicative of and/or predictive of a particular outcome,such as a particular response outcome, such as an objective response(OR) a complete response (CR) or a partial response (PR), or durableresponse, such as an OR or CR or a PR that is durable at 3, 6, 9 monthsor more. In some embodiments, lower or reduced levels or increasedlevels of one or more of such biomarkers (e.g, analytes), such ascompared to a reference value or threshold level, can be associated withthe a response, such as an OR, CR or PR, or any response outcomesdescribed herein, e.g., in Section I.C, optionally a durable response,such as a response that is durable for at least 3 months, 6 months ormore.

In some embodiments, the analyte or biomarker is associated with,correlated to, indicative of and/or predictive of a particular outcome,such as a particular response or durable response outcome, in a subjectthat has been administered a cell therapy, such as with a compositioncontaining genetically engineered cells. In some embodiments, thepresence, expression, level, amount or concentration of one or moreanalyte in a biological sample obtained from a subject prior to theadministration of cell therapy, can be associated with, correlated to,indicative of and/or predictive of a particular outcome, such as aparticular response or durable response outcome. In some embodiments,presence, expression, level, amount or concentration of particularbiomarkers can be correlated to a particular response or durableresponse outcome. In some embodiments, the response outcome can be anyresponse outcomes described herein, e.g., in Section I.C.

In some embodiments, the methods include comparing, individually, thelevel, amount or concentration of the analyte in the sample to athreshold level, thereby determining a likelihood that a subject willachieve a response to the cell therapy. In some embodiments, the methodsinclude selecting a subject who is likely to respond to treatment basedon the results of determining a likelihood that a subject will achieve aresponse to the cell therapy by comparing, individually, the level,amount or concentration of the analyte in the sample to a thresholdlevel. In some embodiments, the methods also include administering thecell therapy to the subject selected for treatment. In some embodiments,if the subject is determined as not likely to achieve a response or adurable response, further comprising administering an additionaltherapeutic agent to the subject.

In some embodiments, the biomarkers (e.g., analytes) include thoseassociated with a response outcome, and/or a durable response. In someembodiments, the biomarkers (e.g. analytes), including parametersthereof, include LDH, ferritin, CRP, D-dimer, Serum Amyloid A1 (SAA-1),IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α and C-X-C motifchemokine 10 (CXCL10).

In some aspects, exemplary analytes or biomarkers that can be assessedor analyzed with respect to assessment of likelihood of response afteradministration of a cell therapy include one or more analyte selectedfrom ferritin, LDH, CXCL10, G-CSF, and IL-10. In some embodiments, forany of the foregoing analytes or biomarkers, the subject is likely toachieve a response if the level, amount or concentration one or more ofthe analyte is below a threshold level and the subject is not likely toachieve a response if the level, amount or concentration one or more ofthe analyte is above a threshold level. In some embodiments, theresponse is or comprises objective response. In some embodiments, theobjective response is or comprises complete response (CR) or partialresponse (PR). In some aspects, reduced levels of ferritin, LDH, CXCL10,G-CSF, and IL-10, in a biological sample from a subject obtained priorto administration of a cell therapy (pre-treatment), can be associatedwith achieving objective response, including complete response (CR) orpartial response (PR).

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 10% or within 5% and/or is within a standarddeviation below the median or mean level, amount or concentration offerritin, LDH, CXCL10, G-CSF, or IL-10 in a biological sample obtainedfrom a group of subjects prior to receiving a cell therapy, wherein eachof the subjects of the group went on to achieve a response afteradministration of a recombinant-receptor-expressing therapeutic cellcomposition for treating the same disease or condition. In someembodiments, the threshold level is within 25%, within 20%, within 15%,within 10% or within 5% and/or is within a standard deviation above themedian or mean level, amount or concentration of ferritin, LDH, CXCL10,G-CSF, or IL-10 in a biological sample obtained from a group of subjectsprior to receiving a cell therapy, wherein each of the subjects of thegroup went on to exhibit stable disease (SD) and/or progressive disease(PD) after administration of a recombinant-receptor-expressingtherapeutic cell composition for treating the same disease or condition.

In some aspects, exemplary analytes or biomarkers that can be assessedor analyzed with respect to assessment of likelihood of durable responseafter administration of a cell therapy include one or more analyteselected from LDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15,IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β. In someembodiments, for any of the foregoing analytes or biomarkers, thesubject is likely to achieve a durable response if the level, amount orconcentration one or more of the analyte is below a threshold level andthe subject is not likely to achieve a durable response if the level,amount or concentration one or more of the analyte is above a thresholdlevel. In some embodiments, the durable response is or comprises acomplete response (CR) or partial response (PR) that is durable for ator greater than 3 months, 4 months, 5 months, or 6 months. In someembodiments, the durable response is or comprises a CR or PR that isdurable for at least 3 months. In some aspects, reduced levels of LDH,ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ,MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β, in a biological sample from asubject obtained prior to administration of a cell therapy(pre-treatment), can be associated with achieving durable response, suchas a CR or PR that is durable for at least 3 months.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 11% or within 5% and/or is within a standarddeviation below the median or mean level, amount or concentration ofLDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α,IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 or MIP-1β in a biological sampleobtained from a group of subjects prior to receiving a cell therapy,wherein each of the subjects of the group went on to achieve a durableresponse after administration of a recombinant-receptor-expressingtherapeutic cell composition for treating the same disease or condition.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 11% or within 5% and/or is within a standarddeviation above the median or mean level, amount or concentration ofLDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α,IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 or MIP-1β in a biological sampleobtained from a group of subjects prior to receiving a cell therapy,wherein each of the subjects of the group did not achieve a durableresponse after administration of a recombinant-receptor-expressingtherapeutic cell composition for treating the same disease or condition.

In some embodiments, the response is durable response, such as a CR orPR that is durable for at least 3 months.

In some embodiments, the threshold level for LDH is at or at about orbelow or below about 600 U/L, 500 U/L, 400 U/L, 300 U/L or 200 U/L.

In some embodiments, exemplary threshold level for ferritin is at or atabout or below or below about 1000 μg/L, 900 μg/L, 800 μg/L, 700 μg/L,600 μg/L, 500 μg/L, 400 μg/L, 300 μg/L or 200 μg/L.

In some embodiments, exemplary threshold level for CRP is at or at aboutor below or below about 20 mg/L, 19 mg/L, 18 mg/L, 17 mg/L, 16 mg/L, 15mg/L, 14 mg/L, 13 mg/L, 12 mg/L, 11 mg/L, 10 mg/L, 9 mg/L, 8 mg/L, 7mg/L, 6 mg/L or 5 mg/L.

In some embodiments, exemplary threshold level for D-dimer is at or atabout or below or below about 1000 μg/L, 900 μg/L, 800 μg/L, 700 μg/L,600 μg/L, 500 μg/L, 400 μg/L, 300 μg/L or 200 μg/L.

In some embodiments, exemplary threshold level for SAA-1 is at or atabout or below or below about 100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60mg/L, 50 mg/L, 40 mg/L, 30 mg/L or 20 mg/L.

In some embodiments, exemplary threshold level for IL-6 is at or atabout or below or below about 6 pg/mL, 5 pg/mL, 4 pg/mL, 3 pg/mL or 2pg/mL.

In some embodiments, exemplary threshold level for IL-10 is at or atabout or below or below about 2 pg/mL, 1 pg/mL, 0.9 pg/mL, 0.8 pg/mL,0.7 pg/mL, 0.6 pg/mL or 0.5 pg/mL.

In some embodiments, exemplary threshold level for IL-15 is at or atabout or below or below about 7 pg/mL, 6 pg/mL, 5 pg/mL, 4 pg/mL or 3pg/mL.

In some embodiments, exemplary threshold level for IL-16 is at or atabout or below or below about 1000 pg/mL, 900 pg/mL, 800 pg/mL, 700pg/mL or 600 pg/mL.

In some embodiments, exemplary threshold level for TNF-α is at or atabout or below or below about 10 pg/mL, 9 pg/mL, 8 pg/mL, 7 pg/mL or 6pg/mL.

In some embodiments, exemplary threshold level for IFN-γ is at or atabout or below or below about 30 pg/mL, 20 pg/mL, 10 pg/mL, 9 pg/mL, 8pg/mL or 7 pg/mL;

In some embodiments, exemplary threshold level for MIP-1α is at or atabout or below or below about 40 pg/mL, 30 pg/mL or 20 pg/mL; and/or

In some embodiments, exemplary threshold level for CXCL-10 is at or atabout or below or below about 1500 pg/mL, 1000 pg/mL, 900 pg/mL, 800pg/mL, 700 pg/mL, 600 pg/mL or 500 pg/mL.

In some aspects, exemplary analytes or biomarkers that can be assessedor analyzed with respect to assessment of likelihood of durable responseafter administration of a cell therapy include one or more analyteselected from ferritin, CRP, LDH, CXCL10, IL-8, IL-10, IL-15, MCP-1,MIP-1β and TNF-α. In some embodiments, for any of the foregoing analytesor biomarkers, the subject is likely to achieve a durable response ifthe level, amount or concentration one or more of the analyte is below athreshold level and the subject is not likely to achieve a durableresponse if the level, amount or concentration one or more of theanalyte is above a threshold level. In some embodiments, the durableresponse is or comprises a complete response (CR) or partial response(PR) that is durable for at or greater than 3 months, 4 months, 5months, or 6 months. In some embodiments, the durable response is orcomprises a CR or PR that is durable for at least 3 months. In someaspects, reduced levels of ferritin, CRP, LDH, CXCL10, IL-8, IL-10,IL-15, MCP-1, MIP-1β and TNF-α, in a biological sample from a subjectobtained prior to administration of a cell therapy (pre-treatment), canbe associated with achieving durable response, such as a CR or PR thatis durable for at least 3 months.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 11% or within 5% and/or is within a standarddeviation below the median or mean level, amount or concentration offerritin, CRP, LDH, CXCL10, IL-8, IL-10, IL-15, MCP-1, MIP-1β or TNF-αin a biological sample obtained from a group of subjects prior toreceiving a cell therapy, wherein each of the subjects of the group wenton to achieve a durable response after administration of arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 11% or within 5% and/or is within a standarddeviation above the median or mean level, amount or concentration offerritin, CRP, LDH, CXCL10, IL-8, IL-10, IL-15, MCP-1, MIP-1β or TNF-αin a biological sample obtained from a group of subjects prior toreceiving a cell therapy, wherein each of the subjects of the group didnot achieve a durable response after administration of arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some aspects, exemplary analytes or biomarkers that can be assessedor analyzed with respect to assessment of likelihood of durable responseafter administration of a cell therapy include one or more analyteselected from hemoglobin and albumin. In some embodiments, for any ofthe foregoing analytes or biomarkers, the subject is likely to achieve adurable response if the level, amount or concentration one or more ofthe analyte is above a threshold level and the subject is not likely toachieve a durable response if the level, amount or concentration one ormore of the analyte is below a threshold level. In some embodiments, thedurable response is or comprises a complete response (CR) or partialresponse (PR) that is durable for at or greater than 3 months, 4 months,5 months, or 6 months. In some embodiments, the durable response is orcomprises a CR or PR that is durable for at least 3 months. In someaspects, elevated levels of hemoglobin and albumin, in a biologicalsample from a subject obtained prior to administration of a cell therapy(pre-treatment), can be associated with achieving durable response, suchas a CR or PR that is durable for at least 3 months.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 11% or within 5% and/or is within a standarddeviation above the median or mean level, amount or concentration ofhemoglobin or albumin in a biological sample obtained from a group ofsubjects prior to receiving a cell therapy, wherein each of the subjectsof the group went on to achieve a durable response after administrationof a recombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 11% or within 5% and/or is within a standarddeviation below the median or mean level, amount or concentration ofhemoglobin or albumin in a biological sample obtained from a group ofsubjects prior to receiving a cell therapy, wherein each of the subjectsof the group did not achieve a durable response after administration ofa recombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

2. Exemplary Biomarkers, Analytes or Parameters Associated with ToxicityOutcomes

In some embodiments, the analyte or biomarker is associated with,correlated to, indicative of and/or predictive of a particular outcome,such as development of a toxicity, in a subject that has beenadministered a cell therapy, such as with a composition containinggenetically engineered cells. In some embodiments, the presence,expression, level, amount or concentration of one or more analyte in abiological sample obtained from a subject prior to the administration ofcell therapy, can be associated with, correlated to, indicative ofand/or predictive of a particular outcome, such as development of atoxicity, such as any toxicity outcomes described herein, e.g., inSection I.D. In some embodiments, presence, expression, level, amount orconcentration of particular biomarkers can be correlated to particularoutcomes or toxicities, e.g., development of NT or CRS. In someembodiments, the toxicity is a toxicity potentially associated with celltherapy, such as any described herein, for example, in Section I.D. Insome embodiments, the toxicity is neurotoxicity (NT) or cytokine releasesyndrome (CRS). In some embodiments, the toxicity is a severe NT orsevere CRS. In some embodiments, the toxicity is grade 2 or higher NT orgrade 2 or higher CRS. In some embodiments, the toxicity is grade 3 orhigher NT or grade 3 or higher CRS.

In some embodiments, the methods include comparing, individually, thelevel, amount or concentration of the analyte in the sample to athreshold level, thereby determining a risk of developing a toxicityafter administration of the cell therapy. In some embodiments, themethods include identifying a subject who has a risk of developing atoxicity after administration of a cell therapy based by comparing,individually, the level, amount or concentration of the analyte in thesample to a threshold level. In some embodiments, the methods alsoinclude following or based on the results of the assessment,administering to the subject the cell therapy, and, optionally, an agentor other treatment capable of treating, preventing, delaying, reducingor attenuating the development or risk of development of a toxicity. Insome embodiments, the methods also involve monitoring the subject forsymptoms of toxicity if the subject is administered a cell therapy andis identified as having a risk of developing a toxicity.

In some embodiments, if the subject is identified as having a risk ofdeveloping a toxicity, one or more of the following steps can beperformed can be administered to the subject: (a) (1) an agent or othertreatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity and (2)the cell therapy, wherein administration of the agent is to beadministered (i) prior to, (ii) within one, two, or three days of, (iii)concurrently with and/or (iv) at first fever following, the initiationof administration of the cell therapy to the subject; and/or (b)administering to the subject a cell therapy at a reduced dose or at adose that is not associated with risk of developing toxicity or severetoxicity, or is not associated with a risk of developing a toxicity orsevere toxicity in a majority of subjects, and/or a majority of subjectshaving a disease or condition that the subject has or is suspected ofhaving, following administration of the cell therapy; and/or (c)administering to the subject a cell therapy in an in-patient settingand/or with admission to the hospital for one or more days, optionallywherein the cell therapy is otherwise to be administered to subjects onan outpatient basis or without admission to the hospital for one or moredays.

In some embodiments, biomarkers or analytes, including parametersthereof, that can be assessed include Lactate dehydrogenase (LDH),ferritin, C-reactive protein (CRP), Interleukin-6 (IL-6), IL-7, IL-8,IL-10, IL-15, IL-16, tumor necrosis factor alpha (TNF-α), interferonalpha 2 (IFN-α2), monocyte chemoattractant protein-1 (MCP-1), macrophageinflammatory protein 1 alpha (MIP-1a), macrophage inflammatory protein 1beta (MIP-1β), Eotaxin, Granulocyte-colony stimulating factor (G-CSF),IL-1 receptor alpha (IL-1Rα), IL-1β, IFN-γ-Inducible Protein 10 (IP-10),perforin, and D-dimer (fibrin degradation product). In some embodiments,the biomarkers (e.g. analytes), including parameters thereof, includeLDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 and MIP-1β.In some embodiments, the biomarkers (e.g. analytes), includingparameters thereof, include ferritin, CRP, D-dimer, IL-6, IL-15, TNF-αand MIP-1α. In some embodiments, the biomarkers (e.g. analytes),including parameters thereof, include ferritin, CRP, IL-10, IL-15,IL-16, TNF-α, or MIP-1β. In some embodiments, elevated levels orincreased levels of one or more of such biomarkers (e.g, biomarkers),such as compared to a reference value or threshold level, can beassociated with the development of neurotoxcity, e.g. severeneurotoxicity or grade 3 or higher or grade 4 or 5 neurotoxicity. Insome embodiments, elevated levels or increased levels of one or more ofsuch biomarkers (e.g, analytes), such as compared to a reference valueor threshold level, can be associated with the development ofneurotoxcity, e.g. severe neurotoxicity or grade 3 or higher or grade 4or 5 neurotoxicity.

In some aspects, exemplary analytes or biomarkers that can be assessedor analyzed with respect to assessment of the risk of developing atoxicity after administration of a cell therapy include one or moreanalyte selected from LDH, Ferritin, C-reactive protein (CRP), IL-6,IL-8, IL-10, TNF-α, IFN-α2, MCP-1 and MIP-1β. In some embodiments, forany of the foregoing analytes or biomarkers, the subject has a risk ofdeveloping a toxicity if the level, amount or concentration one or moreof the analyte is above a threshold level and the subject has a low riskof developing a toxicity if the level, amount or concentration one ormore of the analyte is below a threshold level. In some embodiments, thetoxicity is neurotoxicity. In some aspects, elevated levels of LDH,Ferritin, C-reactive protein (CRP), IL-6, IL-8, IL-10, TNF-α, IFN-α2,MCP-1 and MIP-1β, in a biological sample from a subject obtained priorto administration of a cell therapy (pre-treatment), can be associatedwith a higher risk of developing a neurotoxicity.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 30% or within 5% and/or is within a standarddeviation above the median or mean level, amount or concentration ofLDH, Ferritin, C-reactive protein (CRP), IL-6, IL-8, IL-10, TNF-α,IFN-α2, MCP-1 or MIP-1β in a biological sample obtained from a group ofsubjects prior to receiving a cell therapy, wherein each of the subjectsof the group went on not develop any toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 30% or within 5% and/or is within a standarddeviation below the median or mean level, amount or concentration ofLDH, Ferritin, C-reactive protein (CRP), IL-6, IL-8, IL-10, TNF-α,IFN-α2, MCP-1 or MIP-1β in a biological sample obtained from a group ofsubjects prior to receiving a cell therapy, wherein each of the subjectsof the group went on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments, the toxicity is neurotoxicity.

In some embodiments, exemplary threshold level for LDH is at or at aboutor above or above about 300 U/L, 400 U/L, 500 U/L, 600 U/L or 700 U/L.

In some embodiments, exemplary threshold level for Ferritin is at or atabout or above or above about 500 ng/mL, 600 ng/mL, 700 ng/mL, 800ng/mL, 900 ng/mL, 1000 ng/mL or 1500 ng/mL.

In some embodiments, exemplary threshold level for CRP is at or at aboutor above or above about 20 mg/L, 30 mg/L, 40 mg/L, 50 mg/L, 60 mg/L, 70mg/L or 80 mg/L.

In some embodiments, exemplary threshold level for IL-6 is at or atabout or above or above about 5 pg/mL, 6 pg/mL, 7 pg/mL, 8 pg/mL, 9pg/mL, 10 pg/mL, 20 pg/mL or 30 pg/mL.

In some embodiments, exemplary threshold level for IL-8 is at or atabout or above or above about 8 pg/mL, 9 pg/mL, 10 pg/mL, 20 pg/mL or 30pg/mL.

In some embodiments, exemplary threshold level for IL-10 is at or atabout or above or above about 20 pg/mL, 30 pg/mL, 40 pg/mL, 50 pg/mL, 60pg/mL or 70 pg/mL.

In some embodiments, exemplary threshold level for TNF-α is at or atabout or above or above about 20 pg/mL or 30 pg/mL.

In some embodiments, exemplary threshold level for IFN-α2 is at or atabout or above or above about 40 pg/mL, 50 pg/mL, 60 pg/mL, 70 pg/mL or80 pg/mL.

In some embodiments, exemplary threshold level for MCP-1; and/or is ator at about or above or above about 200 pg/mL or 300 pg/mL.

In some embodiments, exemplary threshold level for MIP-1β is at or atabout or above or above about 40 pg/mL, 50 pg/mL, 60 pg/mL, 70 pg/mL or80 pg/mL.

In some aspects, exemplary analytes or biomarkers that can be assessedor analyzed with respect to assessment of the risk of developing atoxicity after administration of a cell therapy include one or moreanalyte selected from IL-8, IL-10 and CXCL10. In some embodiments, forany of the foregoing analytes or biomarkers, the subject has a risk ofdeveloping a toxicity if the level, amount or concentration one or moreof the analyte is above a threshold level and the subject has a low riskof developing a toxicity if the level, amount or concentration one ormore of the analyte is below a threshold level. In some embodiments, thetoxicity is neurotoxicity. In some embodiments, the toxicity is severeneurotoxicity or a grade 3 or higher neurotoxicity. In some aspects,elevated levels of IL-8, IL-10 and CXCL10, in a biological sample from asubject obtained prior to administration of a cell therapy(pre-treatment), can be associated with a higher risk of developing aneurotoxicity, or a severe neurotoxicity or a grade 3 or higherneurotoxicity.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 30% or within 5% and/or is within a standarddeviation above the median or mean level, amount or concentration ofIL-8, IL-10 or CXCL10 in a biological sample obtained from a group ofsubjects prior to receiving a cell therapy, wherein each of the subjectsof the group went on not develop any toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 30% or within 5% and/or is within a standarddeviation below the median or mean level, amount or concentration ofIL-8, IL-10 or CXCL10 in a biological sample obtained from a group ofsubjects prior to receiving a cell therapy, wherein each of the subjectsof the group went on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some aspects, exemplary analytes or biomarkers or a volumetricmeasure of tumor burden that can be assessed or analyzed with respect toassessment of the risk of developing a toxicity after administration ofa cell therapy include one or more analyte or volumetric measure oftumor burden selected from a sum of the products of diameters (SPD),LDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrin degradationproduct), IL-6, IL-10, IL-15, IL-16 TNF-α, MIP-1α and MIP-1β. In someembodiments, for any of the foregoing analytes or biomarkers orvolumetric measure of tumor burden, the subject has a risk of developinga toxicity if the level, amount or concentration one or more of theanalyte or the volumetric measure of tumor burden is above a thresholdlevel and the subject has a low risk of developing a toxicity if thelevel, amount or concentration one or more of the analyte or thevolumetric measure of tumor burden is below a threshold level. In someembodiments, the toxicity is neurotoxicity. In some aspects, elevatedlevels or measure of a sum of the products of diameters (SPD), LDH,Ferritin, C-reactive protein (CRP), D-dimer (fibrin degradationproduct), IL-6, IL-10, IL-15, IL-16 TNF-α, MIP-1α and MIP-1β, in abiological sample from a subject obtained prior to administration of acell therapy (pre-treatment), can be associated with a higher risk ofdeveloping a neurotoxicity (NT) or a cytokine release syndrome (CRS).

In some embodiments, the one or more analyte or volumetric measure oftumor burden selected from LDH, SPD, IL-10, IL-15, IL-16, TNF-α andMIP-1β, and the toxicity is neurotoxicity In some embodiments, the oneor more analyte or volumetric measure of tumor burden selected from LDH,SPD, CRP, d-dimer, IL-6, IL-15, TNF-α and MIP-1α, and the toxicity isCRS. In some aspects, elevated levels or measure of LDH, SPD, IL-10,IL-15, IL-16, TNF-α and MIP-1β, in a biological sample from a subjectobtained prior to administration of a cell therapy (pre-treatment), canbe associated with a higher risk of developing a neurotoxicity (NT). Insome aspects, elevated levels or measure of LDH, SPD, CRP, d-dimer,IL-6, IL-15, TNF-α and MIP-1α, in a biological sample from a subjectobtained prior to administration of a cell therapy (pre-treatment), canbe associated with a higher risk of developing a cytokine releasesyndrome (CRS).

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 32% or within 5% and/or is within a standarddeviation above the median or mean level, amount or concentration ofLDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrin degradationproduct), IL-6, IL-10, IL-15, IL-16 TNF-α, MIP-1α or MIP-1β, or themedian or mean volumetric measure of tumor burden of a sum of theproducts of diameters (SPD), in a biological sample obtained from agroup of subjects prior to receiving a cell therapy, wherein each of thesubjects of the group went on not develop any toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments, the threshold level is within 25%, within 20%,within 15%, within 32% or within 5% and/or is within a standarddeviation below the median or mean level, amount or concentration ofLDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrin degradationproduct), IL-6, IL-10, IL-15, IL-16 TNF-α, MIP-1α or MIP-1β, or themedian or mean volumetric measure of tumor burden of a sum of theproducts of diameters (SPD), in a biological sample obtained from agroup of subjects prior to receiving a cell therapy, wherein each of thesubjects of the group went on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments, the toxicity is neurotoxicity and exemplarythreshold level for LDH is at or at about or above or above about 300U/L, 400 U/L, 500 U/L or 600 U/L.

In some embodiments, the toxicity is neurotoxicity and exemplarythreshold level for SPD is at or at about or above or above about 30cm², 40 cm², 50 cm², 60 cm², 70 cm², 80 cm² or 90 cm².

In some embodiments, the toxicity is neurotoxicity and exemplarythreshold level for IL-10 is at or at about or above or above about 0.8pg/mL, 0.9 pg/mL, 1 pg/mL, 2 pg/mL, 3 pg/mL or 4 pg/mL.

In some embodiments, the toxicity is neurotoxicity and exemplarythreshold level for IL-15 is at or at about or above or above about 3pg/mL, 4 pg/mL, 5 pg/mL, 6 pg/mL or 7 pg/mL.

In some embodiments, the toxicity is neurotoxicity and exemplarythreshold level for IL-16 is at or at about or above or above about 600pg/mL, 700 pg/mL, 800 pg/mL, 900 pg/mL or 1000 pg/mL.

In some embodiments, the toxicity is neurotoxicity and exemplarythreshold level for TNF-α is at or at about or above or above about 6pg/mL, 7 pg/mL, 8 pg/mL, 9 pg/mL or 10 pg/mL.

In some embodiments, the toxicity is neurotoxicity and exemplarythreshold level for MIP-1β is at or at about or above or above about 70pg/mL, 80 pg/mL, 90 pg/mL or 100 pg/mL.

In some embodiments, the toxicity is CRS and exemplary threshold levelfor LDH is at or at about or above or above about 300 U/L, 400 U/L, 500U/L or 600 U/L.

In some embodiments, the toxicity is CRS the and threshold level for SPDis at or at about or above or above about 20 cm², 30 cm², 40 cm² or 50cm².

In some embodiments, the toxicity is CRS and exemplary threshold levelfor ferritin is at or at about or above or above about 300 ng/mL, 400ng/mL, 500 ng/mL, 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL or 1000ng/mL.

In some embodiments, the toxicity is CRS and exemplary threshold levelfor CRP is at or at about or above or above about 20 mg/L, 30 mg/L or 40mg/L.

In some embodiments, the toxicity is CRS and exemplary threshold levelfor d-dimer is at or at about or above or above about 300 pg/mL, 400pg/mL, 500 pg/mL, 600 pg/mL, 700 pg/mL, 800 pg/mL, 900 pg/mL or 1000pg/mL.

In some embodiments, the toxicity is CRS and exemplary threshold levelfor IL-6 is at or at about or above or above about 2 pg/mL, 3 pg/mL, 4pg/mL, 5 pg/mL, 6 pg/mL, 7 pg/mL, 8 pg/mL or 9 pg/mL.

In some embodiments, the toxicity is CRS and exemplary threshold levelfor IL-15 is at or at about or above or above about 3 pg/mL, 4 pg/mL, 5pg/mL, 6 pg/mL, 7 pg/mL, 8 pg/mL, 9 pg/mL or 10 pg/mL.

In some embodiments, the toxicity is CRS and exemplary threshold levelfor TNF-α is at or at about or above or above about 7 pg/mL, 8 pg/mL, 9pg/mL, 10 pg/mL or 15 pg/mL.

In some embodiments, the toxicity is CRS and exemplary threshold levelfor MIP-1α is at or at about or above or above about 20 pg/mL, 30 pg/mLor 40 pg/mL.

In some embodiments, the biomarker is LDH and in some cases, developmentof toxicity, e.g., CRS or NT, is correlated with the LDH value that isabove a threshold value. In some embodiments, the inflammatory marker isLDH and the threshold value is or is about 300 units per liter, is or isabout 400 units per liter, is or is about 500 units per liter or is oris about 600 units per liter.

In some embodiments, if the level, amount or concentration of thebiomarker (e.g., analyte) in the sample is at or above a threshold levelof the analyte, an agent or other treatment capable of treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity is administered to the subject prior to,within one, two, or three days of, concurrently with and/or at firstfever following, the initiation of administration of the cell therapy tothe subject. Exemplary agents or interventions for use in connectionwith the provided methods to treat, prevent, delay, reduce or attenuatethe risk of developing toxicity are described in Section II.

In some cases, if the level, amount of concentration of the biomarker inthe sample is at or above a threshold level, the cell therapy isadministered to the subject at a reduced dose or at a dose that is notassociated with risk of developing toxicity or severe toxicity, or isnot associated with a risk of developing a toxicity or severe toxicityin a majority of subjects, and/or a majority of subjects having adisease or condition that the subject has or is suspected of having,following administration of the cell therapy. In some cases, if thelevel, amount of concentration of the biomarker in the sample is at orabove a threshold level, the cell therapy is administered to the subjectin an in-patient setting and/or with admission to the hospital for oneor more days, optionally wherein the cell therapy is otherwise to beadministered to subjects on an outpatient basis or without admission tothe hospital for one or more days.

In some embodiments, if the level, amount or concentration of thebiomarker (e.g., analyte) is below a threshold level for the analyte,the cell therapy is administered to the subject, optionally at anon-reduced dose. In some cases, the cells therapy is optionallyadministered on an outpatient basis or without admission to the hospitalfor one or more days. In some embodiments, if the level, amount orconcentration of the analyte, is below a threshold level, theadministration of the cell therapy does not include administering, priorto or concurrently with administering the cell therapy and/or prior tothe development of a sign of symptom of a toxicity other than fever, anagent or treatment capable of treating, preventing, delaying, orattenuating the development of the toxicity; and/or the administrationof the cell therapy is to be or may be administered to the subject on anoutpatient setting and/or without admission of the subject to thehospital overnight or for one or more consecutive days and/or is withoutadmission of the subject to the hospital for one or more days.

In some aspects of the provided methods, a subject is determined to beat risk of developing toxicity (e.g. neurotoxcity, such as severeneurotoxicity or grade 3 or higher neurotoxcity, e.g. grade 4 or 5neurotoxicity and/or CRS, such as severe CRS or grade 3 or higher CRS)by a comparison of the parameter (e.g. concentration, amount, level oractivity) of the biomarker (e.g. analyte) or, individually, each of thebiomarkers (e.g. analytes) to a reference value, such as thresholdlevel, of the corresponding parameter for the biomarker or eachbiomarker. In some embodiments, the comparison indicates whether thesubject is or is not at risk for developing toxicity, e.g.,neurotoxicity such as severe neurotoxicity or grade 3 or higherneurotoxcity, e.g. grade 4 or 5 neurotoxicity and/or CRS, such as severeCRS or grade 3 or higher CRS, and/or indicates a degree of risk fordeveloping said toxicity. In some embodiments, the reference value isone that is a threshold level or cut-off at which there is a goodpredictive value (e.g. accuracy, sensitivity and/or specificity) thatsuch toxicity will occur or is likely to occur either alone or incombination with one or more biomarkers in the panel. In some cases,such reference value, e.g. threshold level, can be or is predeterminedor known prior to performing the method, such as from a plurality ofsubjects previously treated with a cell therapy and assessed for thecorrelation of the parameter of the biomarker or, individually, each ofthe biomarkers in a panel to the presence of a toxic outcome (e.g. thepresence of neurotoxicity such as severe neurotoxicity or grade 3 orhigher neurotoxcity, e.g. grade 4 or 5 neurotoxicity and/or CRS, such assevere CRS or grade 3 or higher CRS).

In some embodiments, a parameter of a biomarker (e.g. LDH, ferritin,CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1 and MIP-1β) that is higheror greater than the reference value, e.g. threshold level, of thecorresponding parameter is associated with a positive prediction of arisk of toxicity (alone or in conjunction with assessment of the otherbiomarkers in the panel). In some embodiments, a parameter of abiomarker that is equal to or lower than the reference value, e.g.threshold level, of the corresponding parameter is associated with anegative prediction of a risk of toxicity (alone or in conjunction withassessment of the other biomarkers in the panel).

In some embodiments, the threshold level is determined based on thelevel, amount, concentration or other measure of the biomarker (e.g.analyte) in the sample positive for the biomarker. In some aspects, thethreshold level is within 25%, within 20%, within 15%, within 10% orwithin 5% of the average level, amount or concentration or measure,and/or is within a standard deviation of the average level, amount orconcentration or measure, of the analyte or parameter in a biologicalsample obtained from a group of subjects prior to receiving arecombinant receptor-expressing therapeutic cell composition, whereineach of the subjects of the group went on to develop a toxicity, e.g.neurotoxicity such as severe neurotoxicity or grade 3 or higherneurotoxcity, e.g. grade 4 or 5 neurotoxicity and/or CRS, such as severeCRS or grade 3 or higher CRS, after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

In some embodiments of any of the provided methods, the biomarker (e.g.analyte) correlates to and/or is predictive of the risk of developingsevere neurotoxicity, such as severe neurotoxicity or grade 3 or higherneurotoxcity, e.g. grade 4 or 5 neurotoxicity and/or severe CRS or grade3 or higher CRS. In some embodiments, the threshold level is within 25%,within 20%, within 15%, within 10% or within 5% of the average level,amount or concentration or measure, and/or is within a standarddeviation of the average level, amount or concentration or measure, ofthe analyte or parameter in a biological sample obtained from a group ofsubjects prior to receiving a recombinant receptor-expressingtherapeutic cell composition, wherein each of the subjects of the groupwent on to develop severe neurotoxicity or grade 3 or higherneurotoxcity, e.g. grade 4 or 5 neurotoxicity and/or severe CRS or grade3 or higher CRS, after receiving a recombinant-receptor-expressingtherapeutic cell composition for treating the same disease or condition.

In some embodiments, the volumetric measure is SPD and in some cases,development of toxicity, e.g., CRS or NT, is correlated with the SPDvalue that is above a threshold value. In some embodiments, thevolumetric measure is SPD, and the threshold value is or is about 30cm², is or is about 40 cm², is or is about 50 cm², is or is about 60cm², or is or is about 70 cm². In some embodiments, the volumetricmeasure is SPD and the threshold value is or is about 30 cm²,is or isabout 40 cm², is or is about 50 cm², is or is about 60 cm², or is or isabout 70 cm².

In some embodiments, the parameter, including volumetric tumormeasurements or is associated with response to the cell therapy, and/ora risk for developing toxicity, e.g., CRS or neurotoxicity (NT).

In some embodiments, the volumetric measure is SPD and the thresholdlevel is or is about 30 cm²,is or is about 40 cm², is or is about 50cm², is or is about 60 cm², or is or is about 70 cm². In someembodiments, the volumetric measure is SPD and the threshold level is oris about 50 cm².

In some embodiments, the analyte is LDH and the threshold level is or isabout 300 units per liter (U/L), is or is about 400 U/L, is or is about500 U/L or is or is about 600 U/L. In some embodiments, the analyte isLDH and the threshold level is or is about 500 U/L.

In some embodiments, the parameter or biomarker is LDH. In someembodiments, the biomarker is LDH and the threshold value is 500 U/L orhigher. In some embodiments, the parameter or biomarker is SPD. In someembodiments, the parameter is SPD, and the threshold value is or isabout 50 cm² or higher. In some embodiments, biomarker or parameters areSPD and LDH, and the threshold values are SPD of 50 cm² or higher andLDH of 500 U/L or higher. In some embodiments, the biomarkers orparameters are associated with increased risk of developing CRS or NT.

In some embodiments, a measurement of the parameter or marker that isabove the threshold value, e.g., SPD of 50 cm² or higher and LDH of 500U/L or higher, are associated with an approximately 2-, 3-, 4-, 5-, 6-,7-, 8-, 9-, 10-fold or more increased risk of developing CRS or NT, suchas any grade CRS or NT. In some embodiments, a measurement of theparameter or marker that is below the threshold value, e.g., SPD oflower than 500 cm² and LDH of lower than 500 U/L, are associated with anapproximately 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold or more decreasedrisk of developing CRS or NT, such as any grade CRS or NT.

In some embodiments, the biomarkers (e.g., analytes), include thoseassociated with increased pharmacokinetic (PK) parameters of the cell,e.g., increased maximum serum concentration of cell (C_(max)) orincreased exposure (e.g., area under the curve (AUC)). In someembodiments, the biomarkers (e.g. analytes), including parametersthereof, include IL-7, IL-15, MIP-1α and TNF-α.

In some embodiments, the parameter is a parameter related to tumorburden, e.g., a measurement of tumor burden. In some aspects, themethods also involve further monitoring the subject for possiblesymptoms of toxicity based on the risk of toxicity determined byassessment of the presence or absence of the biomarker and/or comparisonof the biomarkers to a reference value or threshold level of thebiomarker.

II. Interventions or Agents that Treat or Ameliorate Symptoms ofToxicity

In some embodiments, the provided methods and articles of manufacturecan be used in connection with, or involve or include, one or moreagents or treatments for treating, preventing, delaying, or attenuatingthe development of a toxicity. In some examples, the agent or othertreatment capable of treating, preventing, delaying, or attenuating thedevelopment of a toxicity is administered prior to and/or concurrentlywith administration of a therapeutic cell composition comprising thegenetically engineered cells.

In some embodiments, the agent, e.g., a toxicity-targeting agent, ortreatment capable of treating, preventing, delaying, or attenuating thedevelopment of a toxicity is a steroid, is an antagonist or inhibitor ofa cytokine receptor, such as IL-6 receptor, CD122 receptor (IL-2Rbetareceptor), or CCR2, or is an inhibitor of a cytokine, such as IL-6,MCP-1, IL-10, IFN-γ, IL-8, or IL-18. In some embodiments, the agent isan agonist of a cytokine receptor and/or cytokine, such as TGF-β. Insome embodiments, the agent, e.g., agonist, antagonist or inhibitor, isan antibody or antigen-binding fragment, a small molecule, a protein orpeptide, or a nucleic acid.

In some embodiments, a fluid bolus can be employed as an intervention,such as to treat hypotension associated with CRS. In some embodiments,the target hematocrit levels are >24%. In some embodiments, theintervention includes the use of absorbent resin technology with bloodor plasma filtration. In some cases, the intervention includes dialysis,plasmapheresis, or similar technologies. In some embodiments,vassopressors or acetaminophen can be employed.

In some embodiments, the agent can be administered sequentially,intermittently, or at the same time as or in the same composition as thetherapy, such as cells for adoptive cell therapy. For example, the agentcan be administered before, during, simultaneously with, or afteradministration of the immunotherapy and/or cell therapy.

In some embodiments, the agent is administered at a time as describedherein and in accord with the provided methods, and/or with the providedarticles of manufacture or compositions. In some embodiments, thetoxicity-targeting agent is administered at a time that is within, suchas less than or no more than, 3, 4, 5, 6, 7, 8, 9 or 10 days afterinitiation of the immunotherapy and/or cell therapy. In someembodiments, the toxicity-targeting agent is administered within orwithin about 1 day, 2 days or 3 days after initiation of administrationof the immunotherapy and/or cell therapy.

In some embodiments, the agent, e.g., toxicity-targeting agent, isadministered to a subject after initiation of administration of theimmunotherapy and/or cell therapy at a time at which the subject doesnot exhibit grade 2 or higher CRS or grade 2 or higher neurotoxicity. Insome aspects, the toxicity-targeting agent is administered afterinitiation of administration of the immunotherapy and/or cell therapy ata time at which the subject does not exhibit severe CRS or severeneurotoxicity. Thus, between initiation of administration of theimmunotherapy and/or cell therapy and the toxicity-targeting agent, thesubject is one that does not exhibit grade 2 or higher CRS, such assevere CRS, and/or does not exhibit grade 2 or higher neurotoxicity,such as severe neurotoxicity.

Non-limiting examples of interventions for treating or ameliorating atoxicity, such as severe CRS (sCRS), are described in Table 5. In someembodiments, the intervention includes tocilizumab or othertoxicity-targeting agent as described, which can be at a time in whichthere is a sustained or persistent fever of greater than or about 38° C.or greater than or greater than about 39° C. in the subject. In someembodiments, the fever is sustained in the subject for more than 10hours, more than 12 hours, more than 16 hours, or more than 24 hoursbefore intervention.

TABLE 5 Exemplary Interventions. Symptoms related to CRS SuggestedIntervention Fever of ≥38.3° C. Acetaminophen (12.5 mg/kg) PO/IV up toevery four hours Persistent fever of ≥39° C. for 10 hours that isTocilizumab (8-12 mg/kg) IV unresponsive to acetaminophen Persistentfever of ≥39° C. after tocilizumab Dexamethasone 5-10 mg IV/PO up toevery 6-12 hours with continued fevers Recurrence of symptoms 48 hoursafter initial Tocilizumab (8-12 mg/kg) IV dose of tocilizumabHypotension Fluid bolus, target hematocrit >24% Persistent/recurrenthypotension after initial Tocilizumab (8-12 mg/kg) IV fluid bolus(within 6 hours) Use of low dose pressors for hypotension forDexamethasone 5-10 mg IV/PO up to every longer than 12 hours 6 hourswith continued use of pressors Initiation of higher dose pressors oraddition Dexamethasone 5-10 mg IV/PO up to every of a second pressor forhypotension 6 hours with continued use of pressors Initiation of oxygensupplementation Tocilizumab (8-12 mg/kg) IV Increasing respiratorysupport with concern Dexamethasone 5-10 mg IV/PO up to every forimpending intubation 6 hours with continued use of pressorsRecurrence/Persistence of symptoms for Tocilizumab (8-12 mg/kg) IV whichtocilizumab was given ≥48 hours after initial dose was administered

In some cases, the agent or therapy or intervention, e.g.,toxicity-targeting agent, is administered alone or is administered aspart of a composition or formulation, such as a pharmaceuticalcomposition or formulation, as described herein. Thus, the agent aloneor as part of a pharmaceutical composition can be administeredintravenously or orally, or by any other acceptable known route ofadministration or as described herein.

In some embodiments, the dosage of agent or the frequency ofadministration of the agent in a dosage regimen is reduced compared tothe dosage of the agent or its frequency in a method in which a subjectis treated with the agent after grade 2 or higher CRS or neurotoxicity,such as after severe, e.g., grade 3 or higher, CRS or after severe,e.g., grade 3 or higher neurotoxicity, has developed or been diagnosed(e.g. after physical signs or symptoms of grade 3 or higher CRS orneurotoxicity has manifested). In some embodiments, the dosage of agentor the frequency of administration of the agent in a dosage regimen isreduced compared to the dosage of the agent or its frequency in a methodin which a subject is treated for CRS or neurotoxicity greater than 3days, 4 days, 5 days, 6 days, 1 week, 2 weeks, three weeks, or moreafter administration of the immunotherapy and/or cell therapy. In someembodiments, the dosage is reduced by greater than or greater than about1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold or more. In some embodiments, the dosage isreduced by greater than or about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or more. In some embodiments, the frequency of dosing is reduced,such as the number of daily doses is reduced or the number of days ofdosing is reduced.

A. Steroid

In some embodiments, the agent, e.g., toxicity-targeting agent, thattreats and/or that prevents, delays, or attenuates the development of orrisk for developing a toxicity to an immunotherapy and/or a celltherapy, is a steroid, e.g., corticosteroid. Corticosteroids typicallyinclude glucocorticoids and mineralocorticoids.

Any corticosteroid, e.g., glucocorticoid, can be used in the methodsprovided herein. In some embodiments, glucocorticoids include syntheticand non-synthetic glucocorticoids. Exemplary glucocorticoids include,but are not limited to: alclomethasones, algestones, beclomethasones(e.g. beclomethasone dipropionate), betamethasones (e.g. betamethasone17-valerate, betamethasone sodium acetate, betamethasone sodiumphosphate, betamethasone valerate), budesonides, clobetasols (e.g.clobetasol propionate), clobetasones, clocortolones (e.g. clocortolonepivalate), cloprednols, corticosterones, cortisones and hydrocortisones(e.g. hydrocortisone acetate), cortivazols, deflazacorts, desonides,desoximethasones, dexamethasones (e.g. dexamethasone 21-phosphate,dexamethasone acetate, dexamethasone sodium phosphate), diflorasones(e.g. diflorasone diacetate), diflucortolones, difluprednates,enoxolones, fluazacorts, flucloronides, fludrocortisones (e.g.,fludrocortisone acetate), flumethasones (e.g. flumethasone pivalate),flunisolides, fluocinolones (e.g. fluocinolone acetonide),fluocinonides, fluocortins, fluocortolones, fluorometholones (e.g.fluorometholone acetate), fluperolones (e.g., fluperolone acetate),fluprednidenes, fluprednisolones, flurandrenolides, fluticasones (e.g.fluticasone propionate), formocortals, halcinonides, halobetasols,halometasones, halopredones, hydrocortamates, hydrocortisones (e.g.hydrocortisone 21-butyrate, hydrocortisone aceponate, hydrocortisoneacetate, hydrocortisone buteprate, hydrocortisone butyrate,hydrocortisone cypionate, hydrocortisone hemisuccinate, hydrocortisoneprobutate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, hydrocortisone valerate), loteprednol etabonate,mazipredones, medrysones, meprednisones, methylprednisolones(methylprednisolone aceponate, methylprednisolone acetate,methylprednisolone hemi succinate, methylprednisolone sodium succinate),mometasones (e.g., mometasone furoate), paramethasones (e.g.,paramethasone acetate), prednicarbates, prednisolones (e.g. prednisolone25-diethylaminoacetate, prednisolone sodium phosphate, prednisolone21-hemisuccinate, prednisolone acetate; prednisolone farnesylate,prednisolone hemisuccinate, prednisolone-21 (beta-D-glucuronide),prednisolone metasulphobenzoate, prednisolone steaglate, prednisolonetebutate, prednisolone tetrahydrophthalate), prednisones, prednivals,prednylidenes, rimexolones, tixocortols, triamcinolones (e.g.triamcinolone acetonide, triamcinolone benetonide, triamcinolonehexacetonide, triamcinolone acetonide 21-palmitate, triamcinolonediacetate). These glucocorticoids and the salts thereof are discussed indetail, for example, in Remington's Pharmaceutical Sciences, A. Osol,ed., Mack Pub. Co., Easton, Pa. (16th ed. 1980).

In some examples, the glucocorticoid is selected from among cortisones,dexamethasones, hydrocortisones, methylprednisolones, prednisolones andprednisones. In a particular example, the glucocorticoid isdexamethasone.

In some embodiments, the agent is a corticosteroid and is administeredin an amount that is therapeutically effective to treat, ameliorate orreduce one or more symptoms of a toxicity to an immunotherapy and/or acell therapy, such as CRS or neurotoxicity. In some embodiments,indicators of improvement or successful treatment include determinationof the failure to manifest a relevant score on toxicity grading scale(e.g. CRS or neurotoxicity grading scale), such as a score of less than3, or a change in grading or severity on the grading scale as discussedherein, such as a change from a score of 4 to a score of 3, or a changefrom a score of 4 to a score of 2, 1 or 0.

In some aspects, the corticosteroid is provided in a therapeuticallyeffective dose. Therapeutically effective concentration can bedetermined empirically by testing in known in vitro or in vivo (e.g.animal model) systems. For example, the amount of a selectedcorticosteroid to be administered to ameliorate symptoms or adverseeffects of a toxicity to an immunotherapy and/or a cell therapy, such asCRS or neurotoxicity, can be determined by standard clinical techniques.In addition, animal models can be employed to help identify optimaldosage ranges. The precise dosage, which can be determined empirically,can depend on the particular therapeutic preparation, the regime anddosing schedule, the route of administration and the seriousness of thedisease.

The corticosteroid can be administered in any amount that is effectiveto ameliorate one or more symptoms associated with the toxicity, such aswith the CRS or neurotoxicity. The corticosteroid, e.g., glucocorticoid,can be administered, for example, at an amount between at or about 0.1and 100 mg, per dose, 0.1 to 80 mg, 0.1 to 60 mg, 0.1 to 40 mg, 0.1 to30 mg, 0.1 to 20 mg, 0.1 to 15 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.2 to 40mg, 0.2 to 30 mg, 0.2 to 20 mg, 0.2 to 15 mg, 0.2 to 10 mg, 0.2 to 5 mg,0.4 to 40 mg, 0.4 to 30 mg, 0.4 to 20 mg, 0.4 to 15 mg, 0.4 to 10 mg,0.4 to 5 mg, 0.4 to 4 mg, 1 to 20 mg, 1 to 15 mg or 1 to 10 mg, to a 70kg adult human subject. Typically, the corticosteroid, such as aglucocorticoid is administered at an amount between at or about 0.4 and20 mg, for example, at or about 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.75 mg,0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg or 20mg per dose, to an average adult human subject.

In some embodiments, the corticosteroid can be administered, forexample, at a dosage of at or about 0.001 mg/kg (of the subject), 0.002mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg,0.008 mg/kg, 0.009 mg/kg, 0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.025mg/kg, 0.03 mg/kg, 0.035 mg/kg, 0.04 mg/kg, 0.045 mg/kg, 0.05 mg/kg,0.055 mg/kg, 0.06 mg/kg, 0.065 mg/kg, 0.07 mg/kg, 0.075 mg/kg, 0.08mg/kg, 0.085 mg/kg, 0.09 mg/kg, 0.095 mg/kg, 0.1 mg/kg, 0.15 mg/kg, 0.2mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40 mg/kg, 0.45 mg/kg, 0.50mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg, 0.70 mg/kg, 0.75 mg/kg, 0.80mg/kg, 0.85 mg/kg, 0.90 mg/kg, 0.95 mg/kg, 1 mg/kg, 1.05 mg/kg, 1.1mg/kg, 1.15 mg/kg, 1.20 mg/kg, 1.25 mg/kg, 1.3 mg/kg, 1.35 mg/kg or 1.4mg/kg, to an average adult human subject, typically weighing about 70 kgto 75 kg.

The corticosteroid, or glucocorticoid, for example dexamethasone, can beadministered orally (tablets, liquid or liquid concentrate), PO,intravenously (IV), intramuscularly or by any other known route or routedescribed herein (e.g., with respect to pharmaceutical formulations). Insome aspects, the corticosteroid is administered as a bolus, and inother aspects it may be administered over a period of time.

In some aspects, the glucocorticoid can be administered over a period ofmore than one day, such as over two days, over 3 days, or over 4 or moredays. In some embodiments, the corticosteroid can be administered oneper day, twice per day, or three times or more per day. For example, thecorticosteroid, e.g., dexamethasone, may in some examples beadministered at 10 mg (or equivalent) IV twice a day for three days.

In some embodiments, the dosage of corticosteroid, e.g., glucocorticoid,is administered in successively lower dosages per treatment. Hence, insome such treatment regimes, the dose of corticosteroid is tapered. Forexample, the corticosteroid may be administered at an initial dose (orequivalent dose, such as with reference to dexamethasone) of 4 mg, andupon each successive administration the dose may be lowered, such thatthe dose is 3 mg for the next administration, 2 mg for the nextadministration, and 1 mg for the next administration

Generally, the dose of corticosteroid administered is dependent upon thespecific corticosteroid, as a difference in potency exists betweendifferent corticosteroids. It is typically understood that drugs vary inpotency, and that doses can therefore vary, in order to obtainequivalent effects. Table 6 shows equivalence in terms of potency forvarious glucocorticoids and routes of administration. Equivalent potencyin clinical dosing is well known. Information relating to equivalentsteroid dosing (in a non-chronotherapeutic manner) may be found in theBritish National Formulary (BNF) 37, March 1999.

TABLE 6 Glucocorticoid administration Glucocorticoid (Route) EquivalencyPotency Hydrocortisone (IV or PO) 20 Prednisone 5 Prednisolone (IV orPO) 5 Methylprednisolone sodium succinate (IV) 4 Dexamethasone (IV orPO) 0.5-0.75

Thus, in some embodiments, the steroid is administered in an equivalentdosage amount of from or from about 1.0 mg to 20 mg dexamethasone perday, such as 1.0 mg to 15 mg dexamethasone per day, 1.0 mg to 10 mgdexamethasone per day, 2.0 mg to 8 mg dexamethasone per day, or 2.0 mgto 6.0 mg dexamethasone per day, each inclusive. In some cases, thesteroid is administered in an equivalent dose of at or about 4 mg or ator about 8 mg dexamethasone per day.

In some embodiments, the steroid is administered if fever persists aftertreatment with tocilizumab. For example, in some embodiments,dexamethasone is administered orally or intravenously at a dosage of5-10 mg up to every 6-12 hours with continued fevers. In someembodiments, tocilizumab is administered concurrently with or subsequentto oxygen supplementation.

B. Microglial Cell Inhibitor

In some embodiments, the inhibitor in the combination therapy is aninhibitor of a microglial cell activity. In some embodiments, theadministration of the inhibitor modulates the activity of microglia. Insome embodiments, the inhibitor is an antagonist that inhibits theactivity of a signaling pathway in microglia. In some embodiments, themicroglia inhibitor affects microglial homeostasis, survival, and/orproliferation. In some embodiments, the inhibitor targets the CSF1Rsignaling pathway. In some embodiments, the inhibitor is an inhibitor ofCSF1R. In some embodiments, the inhibitor is a small molecule. In somecases, the inhibitor is an antibody.

In some aspects, administration of the inhibitor results in one or moreeffects selected from an alteration in microglial homeostasis andviability, a decrease or blockade of microglial cell proliferation, areduction or elimination of microglial cells, a reduction in microglialactivation, a reduction in nitric oxide production from microglia, areduction in nitric oxide synthase activity in microglia, or protectionof motor neurons affected by microglial activation. In some embodiments,the agent alters the level of a serum or blood biomarker of CSF1Rinhibition, or a decrease in the level of urinary collagen type 1cross-linked N-telopeptide (NTX) compared to at a time just prior toinitiation of the administration of the inhibitor. In some embodiments,the administration of the agent transiently inhibits the activity ofmicroglia activity and/or wherein the inhibition of microglia activityis not permanent. In some embodiments, the administration of the agenttransiently inhibits the activity of CSF1R and/or wherein the inhibitionof CSF1R activity is not permanent.

In some embodiments, the agent that reduces microglial cell activity isa small molecule, peptide, protein, antibody or antigen-binding fragmentthereof, an antibody mimetic, an aptamer, or a nucleic acid molecule. Insome embodiments, the method involves administration of an inhibitor ofmicroglia activity. In some embodiments, the agent is an antagonist thatinhibits the activity of a signaling pathway in microglia. In someembodiments, the agent that reduces microglial cell activity affectsmicroglial homeostasis, survival, and/or proliferation.

In some embodiments, the agent that reduces microglial cell activationis selected from an anti-inflammatory agent, an inhibitor of NADPHoxidase (NOX2), a calcium channel blocker, a sodium channel blocker,inhibits GM-CSF, inhibits CSF1R, specifically binds CSF-1, specificallybinds IL-34, inhibits the activation of nuclear factor kappa B (NF-κB),activates a CB₂ receptor and/or is a CB₂ agonist, a phosphodiesteraseinhibitor, inhibits microRNA-155 (miR-155), upregulates microRNA-124(miR-124), inhibits nitric oxide production in microglia, inhibitsnitric oxide synthase, or activates the transcription factor NRF2 (alsocalled nuclear factor (erythroid-derived 2)-like 2, or NFE2L2).

In some embodiments, the agent that reduces microglial cell activitytargets CSF1 (also called macrophage colony-stimulating factor MCSF). Insome embodiments, the agent that reduces microglial cell activityaffects MCSF-stimulated phosphorylation of the M-CSF receptor (Pryer etal. Proc Am Assoc Cancer Res, AACR Abstract nr DDT02-2 (2009)). In somecases, the agent that reduces microglial cell activity is MCS110(international patent application publication number WO2014001802;Clinical Trial Study Record Nos.: A1 NCT00757757; NCT02807844;NCT02435680; NCT01643850).

In some embodiments, the agent that reduces microglial cell activity isa small molecule that targets the CSF1 pathway. In some embodiments, theagent is a small molecule that binds CSF1R. In some embodiments, theagent is a small molecule which inhibits CSF1R kinase activity bycompeting with ATP binding to CSF1R kinase. In some embodiments, theagent is a small molecule which inhibits the activation of the CFS1Rreceptor. In some cases, the binding of the CSF-1 ligand to the CSF1R isinhibited. In some embodiments, the agent that reduces microglial cellactivity is any of the inhibitors described in US Patent ApplicationPublication Number US20160032248.

In some embodiments, the agent is a small molecule inhibitor selectedfrom PLX-3397, PLX7486, JNJ-40346527, JNJ28312141, ARRY-382, PLX73086(AC-708), DCC-3014, AZD6495, GW2580, Ki20227, BLZ945, PLX647, PLX5622.In some embodiments, the agent is any of the inhibitors described inConway et al., Proc Natl Acad Sci USA, 102(44):16078-83 (2005); Dagheret al., Journal of Neuroinflammation, 12:139 (2015); Ohno et al., MolCancer Ther. 5(11):2634-43 (2006); von Tresckow et al. Clin Cancer Res.,21(8) (2015); Manthey et al. Mol Cancer Ther. (8(11):3151-61 (2009);Pyonteck et al., Nat Med. 19(10): 1264-1272 (2013); Haegel et al.,Cancer Res AACR Abstract nr 288 (2015); Smith et al., Cancer Res AACRAbstract nr 4889 (2016); Clinical Trial Study Record Nos.: NCT01525602;NCT02734433; NCT02777710; NCT01804530; NCT01597739; NCT01572519;NCT01054014; NCT01316822; NCT02880371; NCT02673736; international patentapplication publication numbers WO2008063888A2, WO2006009755A2, USpatent application publication numbers US20110044998, US 2014/0065141,and US 2015/0119267.

In some embodiments, the agent that reduces microglial cell activity is44(2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide(BLZ945) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is the following compound:

-   -   wherein R1 is an alkyl pyrazole or an alkyl carboxamide, and R2        is a hydroxycycloalkyl or a pharmaceutically acceptable salt        thereof.

In some embodiments, the agent that reduces microglial cell activity is5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)-N-((6-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine,N-[5-[(5-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl]-2-pyridinyl]-6-(trifluoromethyl)-3-pyridinemethanamine)(PLX 3397) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is5-(1H-Pyrrolo[2,3-b]pyridin-3-ylmethyl)-N-[[4-(trifluoromethyl)phenyl]methyl]-2-pyridinaminedihydrochloride (PLX647) or a pharmaceutically acceptable salt thereofor derivatives thereof. In some embodiments, the agent that reducesmicroglial cell activity is the following compound:

-   -   or a pharmaceutically acceptable salt thereof. In some        embodiments, the agent that reduces microglial cell activity is        the following compound:

or a pharmaceutically acceptable salt thereof. In some embodiments, theagent is any of the inhibitors described in U.S. Pat. No. 7,893,075.

In some embodiments, the agent that reduces microglial cell activity is4-cyano-N-[2-(1-cyclohexen-1-yl)-4-[1-[(dimethylamino)acetyl]-4-piperidinyl]phenyl]-1H-imidazole-2-carboxamidemonohydrochloride (JNJ28312141) or a pharmaceutically acceptable saltthereof or derivatives thereof. In some embodiments, the agent is thefollowing compound:

or a pharmaceutically acceptable salt thereof. In some embodiments, theagent is any of the inhibitors described in U.S. Pat. No. 7,645,755.

In some embodiments, the agent that reduces microglial cell activity is1H-Imidazole-2-carboxamide,5-cyano-N-(2-(4,4-dimethyl-1-cyclohexen-1-yl)-6-(tetrahydro-2,2,6,6-tetramethyl-2H-pyran-4-yl)-3-pyridinyl)-,4-Cyano-1H-imidazole-2-carboxylic acidN-(2-(4,4-dimethylcyclohex-1-enyl)-6-(2,2,6,6-tetramethyltetrahydropyran-4-yl)pyridin-3-yl)amide,4-Cyano-N-(2-(4,4-dimethylcyclohex-1-en-1-yl)-6-(2,2,6,6-tetramethyl-tetrahydro-2H-pyran-4-yl)pyridin-3-yl)-1H-imidazole-2-carboxamide(JNJ-40346527) or a pharmaceutically acceptable salt thereof orderivatives thereof. In some embodiments, the agent is the followingcompound:

or a pharmaceutically acceptable salt thereof.

In another embodiment, the agent that reduces microglial cell activityis 5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof (international patentapplication publication number WO2009099553).

In some embodiments, the agent that reduces microglial cell activity is4-(2,4-difluoroanilino)-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoline-3-carboxamide(AZD6495) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activity isN-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N0-[1-(1,3-thiazole-2-yl)ethyl]urea(Ki20227) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activationis an antibody that targets the CSF1 pathway. In some embodiments, theagent is an antibody that binds CSF1R. In some embodiments, theanti-CSF1R antibody blocks CSF1R dimerization. In some embodiments, theanti-CSF1R antibody blocks the CSF1R dimerization interface that isformed by domains D4 and D5 (Ries et al. Cancer Cell 25(6):846-59(2014)). In some cases, the agent is selected from emactuzumab (RG7155;RO5509554), Cabiralizumab (FPA-008), LY-3022855 (IMC-CS4), AMG-820,TG-3003, MCS110, H27K15, 12-2D6, 2-4A5 (Rovida and Sbarba, J Clin CellImmunol. 6: 6 (2015); Clinical Trial Study Record Nos.: NCT02760797;NCT01494688; NCT02323191; NCT01962337; NCT02471716; NCT02526017;NCT01346358; NCT02265536; NCT01444404; NCT02713529, NCT00757757;NCT02807844; NCT02435680; NCT01643850).

In some embodiments, the agent that reduces microglial cell activationis a tetracycline antibiotic. For example, the agent affects IL-lb,IL-6, TNF-α, or iNOS concentration in microglia cells (Yrjänheikki etal. PNAS 95(26): 15769-15774 (1998); Clinical Trial Study Record No:NCT01120899). In some embodiments, the agent is an opioid antagonist(Younger et al. Pain Med. 10(4):663-672 (2009.) In some embodiments, theagent reduces glutamatergic neurotransmission (U.S. Pat. No. 5,527,814).In some embodiments, the agent modulates NFkB signaling (Valera et al J.Neuroinflammation 12:93 (2015); Clinical Trial Study Record No:NCT00231140). In some embodiments, the agent targets cannabinoidreceptors (Ramirez et al. J. Neurosci 25(8):1904-13(2005)). In someembodiments, the agent is selected from minocycline, naloxone, riluzole,lenalidomide, and a cannabinoid (optionally WIN55 or 212-2).

Nitric oxide production from microglia is believed, in some cases, toresult in or increase neurotoxicity. In some embodiments, the agentmodulates or inhibitis nitric oxide production from microglia. In someembodiments, the agent inhibits nitric oxide synthase (NOS). In someembodiments, the NOS inhibitor is Ronopterin (VAS-203), also known as4-amino-tetrahydrobiopterin (4-ABH4). In some embodiments, the NOSinhibitor is cindunistat, A-84643, ONO-1714, L-NOARG, NCX-456, VAS-2381,GW-273629, NXN-462, CKD-712, KD-7040, or guanidinoethyldisulfide. Insome embodiments, the agent is any of the inhibitors described in Minget al., Cell Stem Cell. 2012 Nov. 2; 11(5):620-32.

In some embodiments, the agent blocks T cell trafficking, such as to thecentral nervous system. In some embodiments, blocking T cell traffickingcan reduce or prevent immune cells from crossing blood vessel walls intothe central nervous system, including crossing the blood-brain barrier.In some cases, activated antigen-specific T cells produceproinflammatory cytokines, including IFN-γ and TNF, upon reactivation inthe CNS, leading to activation of resident cells such as microglia andastrocytes. See Kivisakk et al., Neurology. 2009 Jun. 2; 72(22):1922-1930. Thus, in some embodiments, sequestering activated T cellsfrom microglial cells, such as by blocking trafficking and/or inhibitingthe ability of such cells to cross the blood-brain barrier, can reduceor eliminate microglial activation. In some embodiments, the agentinhibits adhesion molecules on immune cells, including T cells. In someembodiments, the agent inhibits an integrin. In some embodiments, theintegrin is alpha-4 integrin. In some embodiments, the agent isnatalizumab (Tysabri®). In some embodiments, the agent modulates a cellsurface receptor. In some embodiments, the agent modulates thesphingosine-1-phosphate (S1P) receptor, such as S1PR1 or S1PR5. In someembodiments, the agent causes the internalization of a cellularreceptor, such as a sphingosine-1-phosphate (S1P) receptor, such asS1PR1 or S1PR5. In some embodiments, the agent is fingolimod (Gilenya®)or ozanimod (RPC-1063).

The transcription factor NRF2 is believed to regulate the anti-oxidantresponse, for example, by turning on genes that contain a cis-actingelement in their promoter region. An example of such an element includesan antioxidant response element (ARE). In some embodiments, the agentactivates NRF2. In some embodiments, activating NRF2 in microglial cellsreduces the microglial cells' responsiveness to IFN and LPS. In someembodiments, activating NRF2 inhibits, slows, or reduces demyelination,axonal loss, neuronal death, and/or oligodendrocyte death. In someembodiments, the agent upregulates the cellular cytoprotective pathwayregulated by NRF2. In some embodiments, the agent that activates NRF2 isdimethyl fumarate (Tecfidera®). In some embodiments, the agent is any ofthe inhibitors described in U.S. Pat. No. 8,399,514. In someembodiments, the agent is any of the inhibitors described in Ming etal., Cell Stem Cell. 2012 Nov. 2; 11(5):620-32.

In some embodiments, the agent that reduces microglial cell activationis(4S,4aS,5aR,12aS)-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide(Minocycline) or a pharmaceutically acceptable salt thereof orderivatives thereof. In some embodiments, the agent is any of thecompounds described in US patent application publication numberUS20100190755. In some embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activationis 3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione (lenalidomide)or a pharmaceutically acceptable salt thereof or derivatives thereof. Insome embodiments, the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activationis4R,4aS,7aR,12bS)-4a,9-dihydroxy-3-prop-2-enyl-2,4,5,6,7a,13-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one(naloxone) or a pharmaceutically acceptable salt thereof or derivativesthereof. In some embodiments, the agent is any of the compoundsdescribed in U.S. Pat. No. 8,247,425. In some embodiments, the agent isthe following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activationis 2-amino-6-(trifluoromethoxy)benzothiazole,6-(trifluoromethoxy)benzo[d]thiazol-2-amine, or6-(trifluoromethoxy)-1,3-benzothiazol-2-amine (riluzole) or apharmaceutically acceptable salt thereof or derivatives thereof asdescribed in U.S. Pat. No. 5,527,8141 In some embodiments, the agent isthe following compound:

or a pharmaceutically acceptable salt thereof.

In some embodiments, the agent that reduces microglial cell activationis a modulator of a signaling pathway in microglia. In some cases, theagent reduces microglia singling. In some embodiments, the agent is aGM-CSF (CSF2) inhibitor. In other embodiments, the agent that reducesmicroglial cell activation is an ion channel blocker. In some specificembodiments, the agent is a calcium channel blocker. For example, insome specific examples, the agent is a dihydropyridine calcium channelblocker. In some embodiments, the agent is a microRNA inhibitor. Forexample, the agent targets miR-155. In some embodiments, the agent thatreduces microglial cell activation is selected from MOR103, Nimodipine,IVIg, and LNA-anti-miR-155 (Butoxsky et al. Ann Neurol., 77(1):75-99(2015) and Sanz et al., Br J Pharmacol. 167(8): 1702-1711 (2012); Winteret al., Ann Clin and Transl Neurol. 2328-9503 (2016); Clinical TrialStudy Record Nos.: NCT01517282, NCT00750867).

In some embodiments, the agent that reduces microglial cell activationis 3-(2-methoxyethyl) 5-propan-2-yl2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate(nimodipine) or a pharmaceutically acceptable salt thereof orderivatives thereof. In some embodiments, the agent is any of theinhibitors described in U.S. Pat. No. 3,799,934. In some embodiments,the agent is the following compound:

or a pharmaceutically acceptable salt thereof.

In some cases, the agent that reduces microglial cell activation isadministered in a form that only affects to central nervous systemand/or does not affect tumor-associated macrophages. In someembodiments, the agent promotes microglia quiescence but does noteliminate or reduce the number of microglia. In some embodiments, themethod involves inhibiting microglia activity specifically in the brainsuch as described in Ponomarev et al., Nature Medicine, (1):64-70 (2011)

Exemplary agents that reduce microglial cell activation, and exemplarydosing regimens for administering such agents, are set forth in Table 7below.

TABLE 7 Exemplary microglia inhibitors and dosage regimens ExemplaryType of Molecular Inhibitor Molecule Target(s) Exemplary DosingRegimen(s) Pexidartinib small molecule CSF1R; c-Kit; 200 mg tablets,twice daily for 28 days; (PLX3397) FLT3 Administer daily as split doseregimen, five dose-levels possible in dose escalation part: 400 mg 5days on 2 days off (intermittent schedule), 400 mg, 600 mg, 800 mg or1000 mg; 1000 mg/day for 2 weeks then 800 mg/day for 22 weeksEmactuzumab monoclonal CSF1R 100-3000 mg once every 2 weeks (RG1755;antibody RO5509554) Cabiralizumab antibody CSF1R Intravenous infusionover 30 minutes (FPA-008) every 2 weeks LY-3022855 monoclonal CSF1R 1.25mg/kg intravenous delivery every 2 (IMC-CS4) antibody weeks for 6 weeksJNJ-40346527 small molecule CSF1R 100 mg twice daily for 12 weeks; 100-1000 mg capsule daily MCS110 antibody MCSF (CSF1) Up to 4 doses of 10mg/kg MCS110 administered intravenously once every 4 weeks starting atDay 1 MORI 03 antibody GM-CSF 6 doses of 0.5-2.0 mg/kg over 70 days IVIgimmunoglobulin Unknown Intravenous infusion of 0.4 g/kg each month for 6months Minocyline small molecule broad spectrum Oral dose of 100 mg ofminocycline antibiotic: IL-1b; twice daily for 24 months IL-6, TNF-a;iNOS Naloxone small molecule Opioid receptors 4.5 mg naltrexonehydrochloride capsules once/day for 8 weeks Lenalidomide/ small moleculeNFkB signaling 100-400 mg daily thalidomide Riluzole small moleculeGlutamate release 50 mg twice daily by microglia Cannabinoids/ smallmolecule cannabinoid Orally 10 mg/kg/day for 6 weeks cannabidiolreceptors (average of 700 mg/day) (e.g. WIN55,212-2) Dimethyl smallmolecule Nrf2 signaling Starting dose of 120 mg taken orally fumaratetwice/day for 7 days. Dose increased to (Tecfidera ®). 240 mg takenorally twice/day thereafter natalizumab antibody alpha-4 integrin 300 mginfused intravenously over (Tysabri ®) one hour, every four weeksfingolimod small molecule S1P receptors, 0.5 mg orally once-daily(Gilenya ®) including S1PR1 ozanimod small molecule S1PR1 and 0.25 mg,0.5 mg, or 1 mg once daily (RPC-1063) S1PR5

C. Other Agents (e.g. Cytokine Targeting Agents)

In some embodiments, the agent, e.g. toxicity-targeting agent, thattreats or ameliorates symptoms of a toxicity of immunotherapy and/or acell therapy, such as CRS or neurotoxicity, is one that targets acytokine, e.g., is an antagonist or inhibitor of a cytokine, such astransforming growth factor beta (TGF-beta), interleukin 6 (IL-6),interleukin 10 (IL-10), IL-2, MIP1β (CCL4), TNF alpha, IL-1, interferongamma (IFN-gamma), or monocyte chemoattractant protein-1 (MCP-1). Insome embodiments, the agent that treats or ameliorates symptoms of atoxicity of an immunotherapy and/or a cell therapy, such as CRS orneurotoxicity, is one that targets (e.g. inhibits or is an antagonistof) a cytokine receptor, such as IL-6 receptor (IL-6R), IL-2 receptor(IL-2R/CD25), MCP-1 (CCL2) receptor (CCR2 or CCR4), a TGF-beta receptor(TGF-beta I, II, or III), IFN-gamma receptor (IFNGR), MIP113 receptor(e.g., CCR5), TNF alpha receptor (e.g., TNFR1), IL-1 receptor(IL1-Rα/IL-1Rβ), or IL-10 receptor (IL-10R).

The amount of a selected agent that treats or ameliorates symptoms of atoxicity of an immunotherapy and/or a cell therapy, such as CRS orneurotoxicity to be administered to ameliorate symptoms or adverseeffects of a toxicity to an immunotherapy and/or a cell therapy, such asCRS or neurotoxicity, can be determined by standard clinical techniques.Exemplary adverse events include, but are not limited to, an increase inalanine aminotransferase, an increase in aspartate aminotransferase,chills, febrile neutropenia, headache, hypotension, left ventriculardysfunction, encephalopathy, hydrocephalus, seizure, and/or tremor.

In some embodiments, the agent is administered in a dosage amount offrom or from about 30 mg to 5000 mg, such as 50 mg to 1000 mg, 50 mg to500 mg, 50 mg to 200 mg, 50 mg to 100 mg, 100 mg to 1000 mg, 100 mg to500 mg, 100 mg to 200 mg, 200 mg to 1000 mg, 200 mg to 500 mg or 500 mgto 1000 mg.

In some embodiments, the agent is administered from or from about 0.5mg/kg to 100 mg/kg, such as from or from about 1 mg/kg to 50 mg/kg, 1mg/kg to 25 mg/kg, 1 mg/kg to 10 mg/kg, 1 mg/kg to 5 mg/kg, 5 mg/kg to100 mg/kg, 5 mg/kg to 50 mg/kg, 5 mg/kg to 25 mg/kg, 5 mg/kg to 10mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, 10 mg/kg to 25mg/kg, 25 mg/kg to 100 mg/kg, 25 mg/kg to 50 mg/kg to 50 mg/kg to 100mg/kg. In some embodiments, the agent is administered in a dosage amountof from or from about 1 mg/kg to 10 mg/kg, 2 mg/kg to 8 mg/kg, 2 mg/kgto 6 mg/kg, 2 mg/kg to 4 mg/kg or 6 mg/kg to 8 mg/kg, each inclusive. Insome aspects, the agent is administered in a dosage amount of at leastor at least about or about 1 mg/kg, 2 mg/kg, 4 mg/kg, 6 mg/kg, 8 mg/kg,10 mg/kg or more. In some embodiments, the agent is administered at adose of 4 mg/kg or 8 mg/kg.

In some embodiments, the agent is administered by injection, e.g.,intravenous or subcutaneous injections, intraocular injection,periocular injection, subretinal injection, intravitreal injection,trans-septal injection, subscleral injection, intrachoroidal injection,intracameral injection, subconjectval injection, subconjuntivalinjection, sub-Tenon's injection, retrobulbar injection, peribulbarinjection, or posterior juxtascleral delivery. In some embodiments, theyare administered by parenteral, intrapulmonary, and intranasal, and, ifdesired for local treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration.

In some embodiments, the amount of the agent is administered about orapproximately twice daily, daily, every other day, three times a week,weekly, every other week or once a month.

In some embodiments, the agent is administered as part of a compositionor formulation, such as a pharmaceutical composition or formulation asdescribed below. Thus, in some cases, the composition comprising theagent is administered as described below. In other aspects, the agent isadministered alone and may be administered by any known acceptable routeof administration or by one described herein, such as with respect tocompositions and pharmaceutical formulations.

In some embodiments, the agent that treats or ameliorates symptoms of atoxicity of the immunotherapy and/or cell therapy, such as CRS orneurotoxicity, is an antibody or antigen binding fragment. In someembodiments, the agent is tocilizumab, siltuximab, sarilumab, olokizumab(CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136),CPSI-2634, ARGX-109, FE301, or FM101.

In some embodiments, the agent is an antagonist or inhibitor of IL-6 orthe IL-6 receptor (IL-6R). In some aspects, the agent is an antibodythat neutralizes IL-6 activity, such as an antibody or antigen-bindingfragment that binds to IL-6 or IL-6R. For example, in some embodiments,the agent is or comprises tocilizumab (atlizumab) or sarilumab,anti-IL-6R antibodies. In some embodiments, the agent is an anti-IL-6Rantibody described in U.S. Pat. No. 8,562,991. In some cases, the agentthat targets IL-6 is an anti-IL-6 antibody, such as siltuximab,elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634,ARGX-109, FE301, FM101, or olokizumab (CDP6038). In some aspects, theagent may neutralize IL-6 activity by inhibiting the ligand-receptorinteractions. The feasibility of this general type of approach has beendemonstrated with a natural occurring receptor antagonist forinterleukin-1. See Harmurn, C. H. et al., Nature (1990) 343:336-340. Insome aspects, the IL-6/IL-6R antagonist or inhibitor is an IL-6 mutein,such as one described in U.S. Pat. No. 5,591,827. In some embodiments,the agent that is an antagonist or inhibitor of IL-6/IL-6R is a smallmolecule, a protein or peptide, or a nucleic acid.

In some embodiments, the agent is tocilizumab. In some embodiments,tocilizumab is administered as an early intervention in accord with theprovided methods, and/or with the provided articles of manufacture orcompositions, at a dosage of from or from about 1 mg/kg to 12 mg/kg,such as at or about 4 mg/kg, 8 mg/kg, or 10 mg/kg. In some embodiments,tocilizumab is administered by intravenous infusion. In someembodiments, tocilizumab is administered for a persistent fever ofgreater than 39° C. lasting 10 hours that is unresponsive toacetaminophen. In some embodiments, a second administration oftocilizumab is provided if symptoms recur after 48 hours of the initialdose.

In some embodiments, the agent is an agonist or stimulator of TGF-β or aTGF-β receptor (e.g., TGF-β receptor I, II, or III). In some aspects,the agent is an antibody that increases TGF-β activity, such as anantibody or antigen-binding fragment that binds to TGF-β or one of itsreceptors. In some embodiments, the agent that is an agonist orstimulator of TGF-β and/or its receptor is a small molecule, a proteinor peptide, or a nucleic acid.

In some embodiments, the agent is an antagonist or inhibitor of MCP-1(CCL2) or a MCP-1 receptor (e.g., MCP-1 receptor CCR2 or CCR4). In someaspects, the agent is an antibody that neutralizes MCP-1 activity, suchas an antibody or antigen-binding fragment that binds to MCP-1 or one ofits receptors (CCR2 or CCR4). In some embodiments, the MCP-1 antagonistor inhibitor is any described in Gong et al. J Exp Med. 1997 Jul. 7;186(1): 131-137 or Shahrara et al. J Immunol 2008; 180:3447-3456. Insome embodiments, the agent that is an antagonist or inhibitor of MCP-1and/or its receptor (CCR2 or CCR4) is a small molecule, a protein orpeptide, or a nucleic acid.

In some embodiments, the agent is an antagonist or inhibitor of IFN-γ oran IFN-γ receptor (IFNGR). In some aspects, the agent is an antibodythat neutralizes IFN-γ activity, such as an antibody or antigen-bindingfragment that binds to IFN-γ or its receptor (IFNGR). In some aspects,the IFN-gamma neutralizing antibody is any described in Dobber et al.Cell Immunol. 1995 February; 160(2):185-92 or Ozmen et al. J Immunol.1993 Apr. 1; 150(7):2698-705. In some embodiments, the agent that is anantagonist or inhibitor of IFN-γ/IFNGR is a small molecule, a protein orpeptide, or a nucleic acid.

In some embodiments, the agent is an antagonist or inhibitor of IL-10 orthe IL-10 receptor (IL-10R). In some aspects, the agent is an antibodythat neutralizes IL-10 activity, such as an antibody or antigen-bindingfragment that binds to IL-10 or IL-10R. In some aspects, the IL-10neutralizing antibody is any described in Dobber et al. Cell Immunol.1995 February; 160(2):185-92 or Hunter et al. J Immunol. 2005 Jun. 1;174(11):7368-75. In some embodiments, the agent that is an antagonist orinhibitor of IL-10/IL-10R is a small molecule, a protein or peptide, ora nucleic acid.

In some embodiments, the agent is an antagonist or inhibitor of IL-1 orthe IL-1 receptor (IL-1R). In some aspects, the agent is an IL-1receptor antagonist, which is a modified form of IL-1R, such as anakinra(see, e.g., Fleischmann et al., (2006) Annals of the rheumatic diseases.65(8):1006-12). In some aspects, the agent is an antibody thatneutralizes IL-1 activity, such as an antibody or antigen-bindingfragment that binds to IL-1 or IL-1R, such as canakinumab (see also EP2277543). In some embodiments, the agent that is an antagonist orinhibitor of IL-1/IL-1R is a small molecule, a protein or peptide, or anucleic acid.

In some embodiments, the agent is an antagonist or inhibitor of a tumornecrosis factor (TNF) or a tumor necrosis factor receptor (TNFR). Insome aspects, the agent is an antibody that blocks TNF activity, such asan antibody or antigen-binding fragment that binds to a TNF, such asTNFα, or its receptor (TNFR, e.g., TNFRp55 or TNFRp75). In some aspects,the agent is selected from among infliximab, adalimumab, certolizumabpegol, golimumab and etanercept. In some embodiments, the agent that isan antagonist or inhibitor of TNF/TNFR is a small molecule, a protein orpeptide, or a nucleic acid. In some embodiments, the agent is a smallmolecule that affects TNF, such as lenalidomide (see, e.g., Muller etal. (1999) Bioorganic & Medicinal Chemistry Letters. 9 (11):1625).

In some embodiments, the agent is an antagonist or inhibitor ofsignaling through the Janus kinase (JAK) and two Signal Transducer andActivator of Transcription (STAT) signaling cascade. JAK/STAT proteinsare common components of cytokine and cytokine receptor signaling. Insome embodiments, the agent that is an antagonist or inhibitor ofJAK/STAT, such as ruxolitinib (see, e.g., Mesa et al. (2012) NatureReviews Drug Discovery. 11(2):103-104), tofacitinib (also known asXeljanz, Jakvinus tasocitinib and CP-690550), Baricitinib (also known asLY-3009104, INCB-28050), Filgotinib (G-146034, GLPG-0634), Gandotinib(LY-2784544), Lestaurtinib (CEP-701), Momelotinib (GS-0387, CYT-387),Pacritinib (SB1518), and Upadacitinib (ABT-494). In some embodiments,the agent is a small molecule, a protein or peptide, or a nucleic acid.

In some embodiments, the agent is a kinase inhibitor. In someembodiments, the agent is an inhibitor of Bruton's tyrosine kinase(BTK). In some embodiments, the inhibitor is or comprises ibrutinib oracalabrutinib (see, e.g., Barrett et al., ASH 58^(th) Annual Meeting SanDiego, Calif. Dec. 3-6, 2016, Abstract 654; Ruella et al., ASH 58thAnnual Meeting San Diego, Calif. Dec. 3-6, 2016, Abstract 2159). In someembodiments, the agent is an inhibitor as described in U.S. Pat. Nos.7,514,444; 8,008,309; 8,476,284; 8,497,277; 8,697,711; 8,703,780;8,735,403; 8,754,090; 8,754,091; 8.957,079; 8,999,999; 9,125,889;9,181,257; or 9,296,753.

In some embodiments, a device, such as absorbent resin technology withblood or plasma filtration, can be used to reduce cytokine levels. Insome embodiments, the device used to reduce cytokine levels is aphysical cytokine absorber, such as an extracorporeal cytokine absorber.In some embodiments, a physical cytokine absorber can be used toeliminate cytokines from the bloodstream in an ex vivo, extracorporealmanner. In some embodiments, the agent is a porous polymer. In someembodiments, the agent is CytoSorb (see, e.g., Basu et al. Indian J CritCare Med. (2014) 18(12): 822-824).

III. Recombinant Antigen Receptors Expressed by the Cells

In some embodiments, the cells for use in or administered in connectionwith the provided methods contain or are engineered to contain anengineered receptor, e.g., an engineered antigen receptor, such as achimeric antigen receptor (CAR), or a T cell receptor (TCR). Alsoprovided are populations of such cells, compositions containing suchcells and/or enriched for such cells, such as in which cells of acertain type such as T cells or CD8⁺ or CD4⁺ cells are enriched orselected. Among the compositions are pharmaceutical compositions andformulations for administration, such as for adoptive cell therapy. Alsoprovided are therapeutic methods for administering the cells andcompositions to subjects, e.g., patients, in accord with the providedmethods, and/or with the provided articles of manufacture orcompositions.

In some embodiments, the cells include one or more nucleic acidsintroduced via genetic engineering, and thereby express recombinant orgenetically engineered products of such nucleic acids. In someembodiments, gene transfer is accomplished by first stimulating thecells, such as by combining it with a stimulus that induces a responsesuch as proliferation, survival, and/or activation, e.g., as measured byexpression of a cytokine or activation marker, followed by transductionof the activated cells, and expansion in culture to numbers sufficientfor clinical applications.

The cells generally express recombinant receptors, such as antigenreceptors including functional non-TCR antigen receptors, e.g., chimericantigen receptors (CARs), and other antigen-binding receptors such astransgenic T cell receptors (TCRs). Also among the receptors are otherchimeric receptors.

A. Chimeric Antigen Receptors (CARs)

In some embodiments of the provided methods and uses, chimericreceptors, such as a chimeric antigen receptors, contain one or moredomains that combine a ligand-binding domain (e.g. antibody or antibodyfragment) that provides specificity for a desired antigen (e.g., tumorantigen) with intracellular signaling domains. In some embodiments, theintracellular signaling domain is a stimulating or an activatingintracellular domain portion, such as a T cell stimulating or activatingdomain, providing a primary activation signal or a primary signal. Insome embodiments, the intracellular signaling domain contains oradditionally contains a costimulatory signaling domain to facilitateeffector functions. In some embodiments, chimeric receptors whengenetically engineered into immune cells can modulate T cell activity,and, in some cases, can modulate T cell differentiation or homeostasis,thereby resulting in genetically engineered cells with improvedlongevity, survival and/or persistence in vivo, such as for use inadoptive cell therapy methods.

Exemplary antigen receptors, including CARs, and methods for engineeringand introducing such receptors into cells, include those described, forexample, in international patent application publication numbersWO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321,WO2013/071154, WO2013/123061 U.S. patent application publication numbersUS2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995,7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319,7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118,and European patent application number EP2537416, and/or those describedby Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila etal. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol.,2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2):160-75. In some aspects, the antigen receptors include a CAR asdescribed in U.S. Pat. No. 7,446,190, and those described inInternational Patent Application Publication No.: WO/2014055668 A1.Examples of the CARs include CARs as disclosed in any of theaforementioned publications, such as WO2014031687, U.S. Pat. Nos.8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190,8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology,10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701;and Brentjens et al., Sci Transl Med. 2013 5(177). See alsoWO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S.Pat. Nos. 7,446,190, and 8,389,282.

The chimeric receptors, such as CARs, generally include an extracellularantigen binding domain, such as a portion of an antibody molecule,generally a variable heavy (V_(H)) chain region and/or variable light(V_(L)) chain region of the antibody, e.g., an scFv antibody fragment.

In some embodiments, the antigen targeted by the receptor is apolypeptide. In some embodiments, it is a carbohydrate or othermolecule. In some embodiments, the antigen is selectively expressed oroverexpressed on cells of the disease or condition, e.g., the tumor orpathogenic cells, as compared to normal or non-targeted cells ortissues. In other embodiments, the antigen is expressed on normal cellsand/or is expressed on the engineered cells.

In some embodiments, the antigen targeted by the receptor is orcomprises selected from among αvβ6 integrin (avb6 integrin), B cellmaturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, alsoknown as CAIX or G250), a cancer-testis antigen, cancer/testis antigen1B (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen(CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19,CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123,CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4),epidermal growth factor protein (EGFR), type III epidermal growth factorreceptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2),epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2(EPHa2), estrogen receptor, Fc receptor like 5 (FCRLS; also known as Fcreceptor homolog 5 or FCRHS), fetal acetylcholine receptor (fetal AchR),a folate binding protein (FBP), folate receptor alpha, ganglioside GD2,O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100),glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPCRSD), Her2/neu(receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbBdimers, Human high molecular weight-melanoma-associated antigen(HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen A1(HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptoralpha(IL-22Rα), IL-13 receptor alpha 2 (IL-13Rα2), kinase insert domainreceptor (kdr), kappa light chain, L1 cell adhesion molecule (L1-CAM),CE7 epitope of L1-CAM, Leucine Rich Repeat Containing 8 Family Member A(LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-A1, MAGE-A3,MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus(CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D)ligands, melan A (MART-1), neural cell adhesion molecule (NCAM),oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME),progesterone receptor, a prostate specific antigen, prostate stem cellantigen (PSCA), prostate specific membrane antigen (PSMA), ReceptorTyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblastglycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72(TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 orgp75), Tyrosinase related protein 2 (TRP2, also known as dopachrometautomerase, dopachrome delta-isomerase or DCT), vascular endothelialgrowth factor receptor (VEGFR), vascular endothelial growth factorreceptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific orpathogen-expressed antigen, or an antigen associated with a universaltag, and/or biotinylated molecules, and/or molecules expressed by HIV,HCV, HBV or other pathogens. Antigens targeted by the receptors in someembodiments include antigens associated with a B cell malignancy, suchas any of a number of known B cell marker. In some embodiments, theantigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21,CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.

In some embodiments the scFv and/or V_(H) domains is derived from FMC63.FMC63 generally refers to a mouse monoclonal IgG1 antibody raisedagainst Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N.R., et al. (1987). Leucocyte typing III. 302). The FMC63 antibodycomprises CDRH1 and H2 set forth in SEQ ID NOS: 38, 39 respectively, andCDRH3 set forth in SEQ ID NOS: 40 or 54 and CDRL1 set forth in SEQ IDNOS: 35 and CDR L2 36 or 55 and CDR L3 sequences 37 or 56. The FMC63antibody comprises the heavy chain variable region (V_(H)) comprisingthe amino acid sequence of SEQ ID NO: 41 and the light chain variableregion (V_(L)) comprising the amino acid sequence of SEQ ID NO: 42. Insome embodiments, the svFv comprises a variable light chain containingthe CDRL1 sequence of SEQ ID NO:35, a CDRL2 sequence of SEQ ID NO:36,and a CDRL3 sequence of SEQ ID NO:37 and/or a variable heavy chaincontaining a CDRH1 sequence of SEQ ID NO:38, a CDRH2 sequence of SEQ IDNO:39, and a CDRH3 sequence of SEQ ID NO:40. In some embodiments, thescFv comprises a variable heavy chain region of FMC63 set forth in SEQID NO:41 and a variable light chain region of FMC63 set forth in SEQ IDNO:42. In some embodiments, the variable heavy and variable light chainare connected by a linker. In some embodiments, the linker is set forthin SEQ ID NO:24. In some embodiments, the scFv comprises, in order, aV_(H), a linker, and a V_(L). In some embodiments, the scFv comprises,in order, a V_(L), a linker, and a V_(H). In some embodiments, the svFcis encoded by a sequence of nucleotides set forth in SEQ ID NO:25 or asequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25.In some embodiments, the scFv comprises the sequence of amino acids setforth in SEQ ID NO:43 or a sequence that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to SEQ ID NO:43.

In some embodiments the scFv is derived from SJ25C1. SJ25C1 is a mousemonoclonal IgG1 antibody raised against Nalm-1 and -16 cells expressingCD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing III.302). The SJ25C1 antibody comprises CDRH1, H2 and H3 set forth in SEQ IDNOS: 47-49, respectively, and CDRL1, L2 and L3 sequences set forth inSEQ ID NOS: 44-46, respectively. The SJ25C1 antibody comprises the heavychain variable region (V_(H)) comprising the amino acid sequence of SEQID NO: 50 and the light chain variable region (V_(L)) comprising theamino acid sequence of SEQ ID NO: 51. In some embodiments, the svFvcomprises a variable light chain containing the CDRL1 sequence of SEQ IDNO:44, a CDRL2 sequence of SEQ ID NO: 45, and a CDRL3 sequence of SEQ IDNO:46 and/or a variable heavy chain containing a CDRH1 sequence of SEQID NO:47, a CDRH2 sequence of SEQ ID NO:48, and a CDRH3 sequence of SEQID NO:49. In some embodiments, the scFv comprises a variable heavy chainregion of SJ25C1 set forth in SEQ ID NO:50 and a variable light chainregion of SJ25C1 set forth in SEQ ID NO:51. In some embodiments, thevariable heavy and variable light chain are connected by a linker. Insome embodiments, the linker is set forth in SEQ ID NO:52. In someembodiments, the scFv comprises, in order, a V_(H), a linker, and aV_(L). In some embodiments, the scFv comprises, in order, a V_(L), alinker, and a V_(H). In some embodiments, the scFv comprises thesequence of amino acids set forth in SEQ ID NO:53 or a sequence thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:53.

In some embodiments, the chimeric antigen receptor includes anextracellular portion containing an antibody or antibody fragment. Insome aspects, the chimeric antigen receptor includes an extracellularportion containing the antibody or fragment and an intracellularsignaling domain. In some embodiments, the antibody or fragment includesan scFv.

In some embodiments, the antibody portion of the recombinant receptor,e.g., CAR, further includes at least a portion of an immunoglobulinconstant region, such as a hinge region, e.g., an IgG4 hinge region,and/or a C_(H)1/C_(L) and/or Fc region. In some embodiments, theconstant region or portion is of a human IgG, such as IgG4 or IgG1. Insome aspects, the portion of the constant region serves as a spacerregion between the antigen-recognition component, e.g., scFv, andtransmembrane domain. The spacer can be of a length that provides forincreased responsiveness of the cell following antigen binding, ascompared to in the absence of the spacer. Exemplary spacers include, butare not limited to, those described in Hudecek et al. (2013) Clin.Cancer Res., 19:3153, international patent application publicationnumber WO2014031687, U.S. Pat. No. 8,822,647 or published app. No.US2014/0271635.

In some embodiments, the constant region or portion is of a human IgG,such as IgG4 or IgG1. In some embodiments, the spacer has the sequenceESKYGPPCPPCP (set forth in SEQ ID NO: 1), and is encoded by the sequenceset forth in SEQ ID NO: 2. In some embodiments, the spacer has thesequence set forth in SEQ ID NO: 3. In some embodiments, the spacer hasthe sequence set forth in SEQ ID NO: 4. In some embodiments, theconstant region or portion is of IgD. In some embodiments, the spacerhas the sequence set forth in SEQ ID NO: 5. In some embodiments, thespacer has a sequence of amino acids that exhibits at least 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to any of SEQ ID NOS: 1, 3, 4 or 5. In someembodiments, the spacer has the sequence set forth in SEQ ID NOS: 26-34.In some embodiments, the spacer has a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS:26-34.

In some embodiments, the antigen receptor comprises an intracellulardomain linked directly or indirectly to the extracellular domain. Insome embodiments, the chimeric antigen receptor includes a transmembranedomain linking the extracellular domain and the intracellular signalingdomain. In some embodiments, the intracellular signaling domaincomprises an ITAM. For example, in some aspects, the antigen recognitiondomain (e.g. extracellular domain) generally is linked to one or moreintracellular signaling components, such as signaling components thatmimic activation through an antigen receptor complex, such as a TCRcomplex, in the case of a CAR, and/or signal via another cell surfacereceptor. In some embodiments, the chimeric receptor comprises atransmembrane domain linked or fused between the extracellular domain(e.g. scFv) and intracellular signaling domain. Thus, in someembodiments, the antigen-binding component (e.g., antibody) is linked toone or more transmembrane and intracellular signaling domains.

In one embodiment, a transmembrane domain that naturally is associatedwith one of the domains in the receptor, e.g., CAR, is used. In someinstances, the transmembrane domain is selected or modified by aminoacid substitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex.

The transmembrane domain in some embodiments is derived either from anatural or from a synthetic source. Where the source is natural, thedomain in some aspects is derived from any membrane-bound ortransmembrane protein. Transmembrane regions include those derived from(i.e. comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.Alternatively the transmembrane domain in some embodiments is synthetic.In some aspects, the synthetic transmembrane domain comprisespredominantly hydrophobic residues such as leucine and valine. In someaspects, a triplet of phenylalanine, tryptophan and valine will be foundat each end of a synthetic transmembrane domain. In some embodiments,the linkage is by linkers, spacers, and/or transmembrane domain(s). Insome aspects, the transmembrane domain contains a transmembrane portionof CD28.

In some embodiments, the extracellular domain and transmembrane domaincan be linked directly or indirectly. In some embodiments, theextracellular domain and transmembrane are linked by a spacer, such asany described herein. In some embodiments, the receptor containsextracellular portion of the molecule from which the transmembranedomain is derived, such as a CD28 extracellular portion.

Among the intracellular signaling domains are those that mimic orapproximate a signal through a natural antigen receptor, a signalthrough such a receptor in combination with a costimulatory receptor,and/or a signal through a costimulatory receptor alone. In someembodiments, a short oligo- or polypeptide linker, for example, a linkerof between 2 and 10 amino acids in length, such as one containingglycines and serines, e.g., glycine-serine doublet, is present and formsa linkage between the transmembrane domain and the cytoplasmic signalingdomain of the CAR.

T cell activation is in some aspects described as being mediated by twoclasses of cytoplasmic signaling sequences: those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences), and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences). In some aspects, theCAR includes one or both of such signaling components.

The receptor, e.g., the CAR, generally includes at least oneintracellular signaling component or components. In some aspects, theCAR includes a primary cytoplasmic signaling sequence that regulatesprimary activation of the TCR complex. Primary cytoplasmic signalingsequences that act in a stimulatory manner may contain signaling motifswhich are known as immunoreceptor tyrosine-based activation motifs orITAMs. Examples of ITAM containing primary cytoplasmic signalingsequences include those derived from CD3 zeta chain, FcR gamma, CD3gamma, CD3 delta and CD3 epsilon. In some embodiments, cytoplasmicsignaling molecule(s) in the CAR contain(s) a cytoplasmic signalingdomain, portion thereof, or sequence derived from CD3 zeta.

In some embodiments, the receptor includes an intracellular component ofa TCR complex, such as a TCR CD3 chain that mediates T-cell activationand cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, theantigen-binding portion is linked to one or more cell signaling modules.In some embodiments, cell signaling modules include CD3 transmembranedomain, CD3 intracellular signaling domains, and/or other CDtransmembrane domains. In some embodiments, the receptor, e.g., CAR,further includes a portion of one or more additional molecules such asFc receptor α, CD8, CD4, CD25, or CD16. For example, in some aspects,the CAR or other chimeric receptor includes a chimeric molecule betweenCD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.

In some embodiments, upon ligation of the CAR or other chimericreceptor, the cytoplasmic domain or intracellular signaling domain ofthe receptor activates at least one of the normal effector functions orresponses of the immune cell, e.g., T cell engineered to express theCAR. For example, in some contexts, the CAR induces a function of a Tcell such as cytolytic activity or T-helper activity, such as secretionof cytokines or other factors. In some embodiments, a truncated portionof an intracellular signaling domain of an antigen receptor component orcostimulatory molecule is used in place of an intact immunostimulatorychain, for example, if it transduces the effector function signal. Insome embodiments, the intracellular signaling domain or domains includethe cytoplasmic sequences of the T cell receptor (TCR), and in someaspects also those of co-receptors that in the natural context act inconcert with such receptors to initiate signal transduction followingantigen receptor engagement.

In the context of a natural TCR, full activation generally requires notonly signaling through the TCR, but also a costimulatory signal. Thus,in some embodiments, to promote full activation, a component forgenerating secondary or co-stimulatory signal is also included in theCAR. In other embodiments, the CAR does not include a component forgenerating a costimulatory signal. In some aspects, an additional CAR isexpressed in the same cell and provides the component for generating thesecondary or costimulatory signal.

In some embodiments, the chimeric antigen receptor contains anintracellular domain of a T cell costimulatory molecule. In someembodiments, the CAR includes a signaling domain and/or transmembraneportion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DAP10,and ICOS. In some aspects, the same CAR includes both the activating andcostimulatory components. In some embodiments, the chimeric antigenreceptor contains an intracellular domain derived from a T cellcostimulatory molecule or a functional variant thereof, such as betweenthe transmembrane domain and intracellular signaling domain. In someaspects, the T cell costimulatory molecule is CD28 or 41BB.

In some embodiments, the activating domain is included within one CAR,whereas the costimulatory component is provided by another CARrecognizing another antigen. In some embodiments, the CARs includeactivating or stimulatory CARs, costimulatory CARs, both expressed onthe same cell (see WO2014/055668). In some aspects, the cells includeone or more stimulatory or activating CAR and/or a costimulatory CAR. Insome embodiments, the cells further include inhibitory CARs (iCARs, seeFedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such asa CAR recognizing an antigen other than the one associated with and/orspecific for the disease or condition whereby an activating signaldelivered through the disease-targeting CAR is diminished or inhibitedby binding of the inhibitory CAR to its ligand, e.g., to reduceoff-target effects.

In certain embodiments, the intracellular signaling domain comprises aCD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta)intracellular domain. In some embodiments, the intracellular signalingdomain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9)co-stimulatory domains, linked to a CD3 zeta intracellular domain.

In some embodiments, the CAR encompasses one or more, e.g., two or more,costimulatory domains and an activation domain, e.g., primary activationdomain, in the cytoplasmic portion. Exemplary CARs include intracellularcomponents of CD3-zeta, CD28, and 4-1BB.

In some embodiments, the antigen receptor further includes a markerand/or cells expressing the CAR or other antigen receptor furtherincludes a surrogate marker, such as a cell surface marker, which may beused to confirm transduction or engineering of the cell to express thereceptor. In some aspects, the marker includes all or part (e.g.,truncated form) of CD34, a NGFR, or epidermal growth factor receptor,such as truncated version of such a cell surface receptor (e.g., tEGFR).In some embodiments, the nucleic acid encoding the marker is operablylinked to a polynucleotide encoding for a linker sequence, such as acleavable linker sequence, e.g., T2A. For example, a marker, andoptionally a linker sequence, can be any as disclosed in publishedpatent application No. WO2014031687. For example, the marker can be atruncated EGFR (tEGFR) that is, optionally, linked to a linker sequence,such as a T2A cleavable linker sequence.

An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises thesequence of amino acids set forth in SEQ ID NO: 7 or 16 or a sequence ofamino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQID NO: 7 or 16. An exemplary T2A linker sequence comprises the sequenceof amino acids set forth in SEQ ID NO: 6 or 17 or a sequence of aminoacids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ IDNO: 6 or 17.

In some embodiments, the marker is a molecule, e.g., cell surfaceprotein, not naturally found on T cells or not naturally found on thesurface of T cells, or a portion thereof. In some embodiments, themolecule is a non-self molecule, e.g., non-self protein, i.e., one thatis not recognized as “self” by the immune system of the host into whichthe cells will be adoptively transferred.

In some embodiments, the marker serves no therapeutic function and/orproduces no effect other than to be used as a marker for geneticengineering, e.g., for selecting cells successfully engineered. In otherembodiments, the marker may be a therapeutic molecule or moleculeotherwise exerting some desired effect, such as a ligand for a cell tobe encountered in vivo, such as a costimulatory or immune checkpointmolecule to enhance and/or dampen responses of the cells upon adoptivetransfer and encounter with ligand.

In some cases, CARs are referred to as first, second, and/or thirdgeneration CARs. In some aspects, a first generation CAR is one thatsolely provides a CD3-chain induced signal upon antigen binding; in someaspects, a second-generation CARs is one that provides such a signal andcostimulatory signal, such as one including an intracellular signalingdomain from a costimulatory receptor such as CD28 or CD137; in someaspects, a third generation CAR is one that includes multiplecostimulatory domains of different costimulatory receptors.

For example, in some embodiments, the CAR contains an antibody, e.g., anantibody fragment, a transmembrane domain that is or contains atransmembrane portion of CD28 or a functional variant thereof, and anintracellular signaling domain containing a signaling portion of CD28 orfunctional variant thereof and a signaling portion of CD3 zeta orfunctional variant thereof. In some embodiments, the CAR contains anantibody, e.g., antibody fragment, a transmembrane domain that is orcontains a transmembrane portion of CD28 or a functional variantthereof, and an intracellular signaling domain containing a signalingportion of a 4-1BB or functional variant thereof and a signaling portionof CD3 zeta or functional variant thereof. In some such embodiments, thereceptor further includes a spacer containing a portion of an Igmolecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4hinge, such as a hinge-only spacer.

In some embodiments, the transmembrane domain of the recombinantreceptor, e.g., the CAR, is or includes a transmembrane domain of humanCD28 (e.g. Accession No. P01747.1) or variant thereof, such as atransmembrane domain that comprises the sequence of amino acids setforth in SEQ ID NO: 8 or a sequence of amino acids that exhibits atleast 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to SEQ ID NO: 8; in some embodiments,the transmembrane-domain containing portion of the recombinant receptorcomprises the sequence of amino acids set forth in SEQ ID NO: 9 or asequence of amino acids having at least at or about 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity thereto.

In some embodiments, the intracellular signaling component(s) of therecombinant receptor, e.g. the CAR, contains an intracellularcostimulatory signaling domain of human CD28 or a functional variant orportion thereof, such as a domain with an LL to GG substitution atpositions 186-187 of a native CD28 protein. For example, theintracellular signaling domain can comprise the sequence of amino acidsset forth in SEQ ID NO: 10 or 11 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11. Insome embodiments, the intracellular domain comprises an intracellularcostimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1)or functional variant or portion thereof, such as the sequence of aminoacids set forth in SEQ ID NO: 12 or a sequence of amino acids thatexhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.

In some embodiments, the intracellular signaling domain of therecombinant receptor, e.g. the CAR, comprises a human CD3 zetastimulatory signaling domain or functional variant thereof, such as an112 AA cytoplasmic domain of isoform 3 of human CD3 (Accession No.:P20963.2) or a CD3 zeta signaling domain as described in U.S. Pat. No.7,446,190 or U.S. Pat. No. 8,911,993. For example, in some embodiments,the intracellular signaling domain comprises the sequence of amino acidsas set forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acidsthat exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14or 15.

In some aspects, the spacer contains only a hinge region of an IgG, suchas only a hinge of IgG4 or IgG1, such as the hinge only spacer set forthin SEQ ID NO: 1. In other embodiments, the spacer is or contains an Ighinge, e.g., an IgG4-derived hinge, optionally linked to a C_(H)2 and/orC_(H)3 domains. In some embodiments, the spacer is an Ig hinge, e.g., anIgG4 hinge, linked to C_(H)2 and C_(H)3 domains, such as set forth inSEQ ID NO: 4. In some embodiments, the spacer is an Ig hinge, e.g., anIgG4 hinge, linked to a C_(H)3 domain only, such as set forth in SEQ IDNO: 3. In some embodiments, the spacer is or comprises a glycine-serinerich sequence or other flexible linker such as known flexible linkers.

For example, in some embodiments, the CAR includes an antibody such asan antibody fragment, including scFvs, a spacer, such as a spacercontaining a portion of an immunoglobulin molecule, such as a hingeregion and/or one or more constant regions of a heavy chain molecule,such as an Ig-hinge containing spacer, a transmembrane domain containingall or a portion of a CD28-derived transmembrane domain, a CD28-derivedintracellular signaling domain, and a CD3 zeta signaling domain. In someembodiments, the CAR includes an antibody or fragment, such as scFv, aspacer such as any of the Ig-hinge containing spacers, a CD28-derivedtransmembrane domain, a 4-1BB-derived intracellular signaling domain,and a CD3 zeta-derived signaling domain.

In some embodiments, nucleic acid molecules encoding such CAR constructsfurther includes a sequence encoding a T2A ribosomal skip element and/ora tEGFR sequence, e.g., downstream of the sequence encoding the CAR. Insome embodiments, the sequence encodes a T2A ribosomal skip element setforth in SEQ ID NO: 6 or 17, or a sequence of amino acids that exhibitsat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity to SEQ ID NO: 6 or 17. In someembodiments, T cells expressing an antigen receptor (e.g. CAR) can alsobe generated to express a truncated EGFR (EGFRt) as a non-immunogenicselection epitope (e.g. by introduction of a construct encoding the CARand EGFRt separated by a T2A ribosome switch to express two proteinsfrom the same construct), which then can be used as a marker to detectsuch cells (see e.g. U.S. Pat. No. 8,802,374). In some embodiments, thesequence encodes an tEGFR sequence set forth in SEQ ID NO: 7 or 16, or asequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to SEQ ID NO: 7 or 16. In some cases, the peptide, such as T2A,can cause the ribosome to skip (ribosome skipping) synthesis of apeptide bond at the C-terminus of a 2A element, leading to separationbetween the end of the 2A sequence and the next peptide downstream (see,for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) anddeFelipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known.Examples of 2A sequences that can be used in the methods and nucleicacids disclosed herein, without limitation, 2A sequences from thefoot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 21), equine rhinitisA virus (E2A, e.g., SEQ ID NO: 20), Thosea asigna virus (T2A, e.g., SEQID NO: 6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 18 or19) as described in U.S. Patent Publication No. 20070116690.

The recombinant receptors, such as CARs, expressed by the cellsadministered to the subject generally recognize or specifically bind toa molecule that is expressed in, associated with, and/or specific forthe disease or condition or cells thereof being treated. Upon specificbinding to the molecule, e.g., antigen, the receptor generally deliversan immunostimulatory signal, such as an ITAM-transduced signal, into thecell, thereby promoting an immune response targeted to the disease orcondition. For example, in some embodiments, the cells express a CARthat specifically binds to an antigen expressed by a cell or tissue ofthe disease or condition or associated with the disease or condition.

B. T Cell Receptors (TCRs)

In some embodiments, engineered cells, such as T cells, used inconnection with the provided methods, uses, articles of manufacture orcompositions are cells that express a T cell receptor (TCR) orantigen-binding portion thereof that recognizes an peptide epitope or Tcell epitope of a target polypeptide, such as an antigen of a tumor,viral or autoimmune protein.

In some embodiments, a “T cell receptor” or “TCR” is a molecule thatcontains a variable α and β chains (also known as TCRα and TCRβ,respectively) or a variable γ and δ chains (also known as TCRα and TCRβ,respectively), or antigen-binding portions thereof, and which is capableof specifically binding to a peptide bound to an MHC molecule. In someembodiments, the TCR is in the αβ form. Typically, TCRs that exist in αβand γδ forms are generally structurally similar, but T cells expressingthem may have distinct anatomical locations or functions. A TCR can befound on the surface of a cell or in soluble form. Generally, a TCR isfound on the surface of T cells (or T lymphocytes) where it is generallyresponsible for recognizing antigens bound to major histocompatibilitycomplex (MHC) molecules.

Unless otherwise stated, the term “TCR” should be understood toencompass full TCRs as well as antigen-binding portions orantigen-binding fragments thereof. In some embodiments, the TCR is anintact or full-length TCR, including TCRs in the αβ form or γδ form. Insome embodiments, the TCR is an antigen-binding portion that is lessthan a full-length TCR but that binds to a specific peptide bound in anMHC molecule, such as binds to an MHC-peptide complex. In some cases, anantigen-binding portion or fragment of a TCR can contain only a portionof the structural domains of a full-length or intact TCR, but yet isable to bind the peptide epitope, such as MHC-peptide complex, to whichthe full TCR binds. In some cases, an antigen-binding portion containsthe variable domains of a TCR, such as variable α chain and variable βchain of a TCR, sufficient to form a binding site for binding to aspecific MHC-peptide complex. Generally, the variable chains of a TCRcontain complementarity determining regions involved in recognition ofthe peptide, MHC and/or MHC-peptide complex.

In some embodiments, the variable domains of the TCR containhypervariable loops, or complementarity determining regions (CDRs),which generally are the primary contributors to antigen recognition andbinding capabilities and specificity. In some embodiments, a CDR of aTCR or combination thereof forms all or substantially all of theantigen-binding site of a given TCR molecule. The various CDRs within avariable region of a TCR chain generally are separated by frameworkregions (FRs), which generally display less variability among TCRmolecules as compared to the CDRs (see, e.g., Jores et al., Proc. Nat'lAcad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988;see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In someembodiments, CDR3 is the main CDR responsible for antigen binding orspecificity, or is the most important among the three CDRs on a givenTCR variable region for antigen recognition, and/or for interaction withthe processed peptide portion of the peptide-MHC complex. In somecontexts, the CDR1 of the alpha chain can interact with the N-terminalpart of certain antigenic peptides. In some contexts, CDR1 of the betachain can interact with the C-terminal part of the peptide. In somecontexts, CDR2 contributes most strongly to or is the primary CDRresponsible for the interaction with or recognition of the MHC portionof the MHC-peptide complex. In some embodiments, the variable region ofthe (3-chain can contain a further hypervariable region (CDR4 or HVR4),which generally is involved in superantigen binding and not antigenrecognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).

In some embodiments, a TCR also can contain a constant domain, atransmembrane domain and/or a short cytoplasmic tail (see, e.g., Janewayet al., Immunobiology: The Immune System in Health and Disease, 3rd Ed.,Current Biology Publications, p. 4:33, 1997). In some aspects, eachchain of the TCR can possess one N-terminal immunoglobulin variabledomain, one immunoglobulin constant domain, a transmembrane region, anda short cytoplasmic tail at the C-terminal end. In some embodiments, aTCR is associated with invariant proteins of the CD3 complex involved inmediating signal transduction.

In some embodiments, a TCR chain contains one or more constant domain.For example, the extracellular portion of a given TCR chain (e.g.,α-chain or β-chain) can contain two immunoglobulin-like domains, such asa variable domain (e.g., Vα or Vβ; typically amino acids 1 to 116 basedon Kabat numbering Kabat et al., “Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services, Public Health ServiceNational Institutes of Health, 1991, 5th ed.) and a constant domain(e.g., α-chain constant domain or Cα, typically positions 117 to 259 ofthe chain based on Kabat numbering or β chain constant domain or C_(β),typically positions 117 to 295 of the chain based on Kabat) adjacent tothe cell membrane. For example, in some cases, the extracellular portionof the TCR formed by the two chains contains two membrane-proximalconstant domains, and two membrane-distal variable domains, whichvariable domains each contain CDRs. The constant domain of the TCR maycontain short connecting sequences in which a cysteine residue forms adisulfide bond, thereby linking the two chains of the TCR. In someembodiments, a TCR may have an additional cysteine residue in each ofthe α and β chains, such that the TCR contains two disulfide bonds inthe constant domains.

In some embodiments, the TCR chains contain a transmembrane domain. Insome embodiments, the transmembrane domain is positively charged. Insome cases, the TCR chain contains a cytoplasmic tail. In some cases,the structure allows the TCR to associate with other molecules like CD3and subunits thereof. For example, a TCR containing constant domainswith a transmembrane region may anchor the protein in the cell membraneand associate with invariant subunits of the CD3 signaling apparatus orcomplex. The intracellular tails of CD3 signaling subunits (e.g. CD3γ,CD3δ, CD3ε and CD3ζ chains) contain one or more immunoreceptortyrosine-based activation motif or ITAM that are involved in thesignaling capacity of the TCR complex.

In some embodiments, the TCR may be a heterodimer of two chains α and β(or optionally γ and δ) or it may be a single chain TCR construct. Insome embodiments, the TCR is a heterodimer containing two separatechains (α and β chains or γ and δ chains) that are linked, such as by adisulfide bond or disulfide bonds.

In some embodiments, the TCR can be generated from a known TCRsequence(s), such as sequences of Vα,β chains, for which a substantiallyfull-length coding sequence is readily available. Methods for obtainingfull-length TCR sequences, including V chain sequences, from cellsources are well known. In some embodiments, nucleic acids encoding theTCR can be obtained from a variety of sources, such as by polymerasechain reaction (PCR) amplification of TCR-encoding nucleic acids withinor isolated from a given cell or cells, or synthesis of publiclyavailable TCR DNA sequences.

In some embodiments, the TCR is obtained from a biological source, suchas from cells such as from a T cell (e.g. cytotoxic T cell), T-cellhybridomas or other publicly available source. In some embodiments, theT-cells can be obtained from in vivo isolated cells. In someembodiments, the TCR is a thymically selected TCR. In some embodiments,the TCR is a neoepitope-restricted TCR. In some embodiments, the T-cellscan be a cultured T-cell hybridoma or clone. In some embodiments, theTCR or antigen-binding portion thereof or antigen-binding fragmentthereof can be synthetically generated from knowledge of the sequence ofthe TCR.

In some embodiments, the TCR is generated from a TCR identified orselected from screening a library of candidate TCRs against a targetpolypeptide antigen, or target T cell epitope thereof. TCR libraries canbe generated by amplification of the repertoire of Vα and Vβ from Tcells isolated from a subject, including cells present in PBMCs, spleenor other lymphoid organ. In some cases, T cells can be amplified fromtumor-infiltrating lymphocytes (TILs). In some embodiments, TCRlibraries can be generated from CD4⁺ or CD8⁺ cells. In some embodiments,the TCRs can be amplified from a T cell source of a normal of healthysubject, i.e. normal TCR libraries. In some embodiments, the TCRs can beamplified from a T cell source of a diseased subject, i.e. diseased TCRlibraries. In some embodiments, degenerate primers are used to amplifythe gene repertoire of Vα and Vβ, such as by RT-PCR in samples, such asT cells, obtained from humans. In some embodiments, scTv libraries canbe assembled from naïve Vα and Vβ libraries in which the amplifiedproducts are cloned or assembled to be separated by a linker. Dependingon the source of the subject and cells, the libraries can be HLAallele-specific. Alternatively, in some embodiments, TCR libraries canbe generated by mutagenesis or diversification of a parent or scaffoldTCR molecule. In some aspects, the TCRs are subjected to directedevolution, such as by mutagenesis, e.g., of the α or β chain. In someaspects, particular residues within CDRs of the TCR are altered. In someembodiments, selected TCRs can be modified by affinity maturation. Insome embodiments, antigen-specific T cells may be selected, such as byscreening to assess CTL activity against the peptide. In some aspects,TCRs, e.g. present on the antigen-specific T cells, may be selected,such as by binding activity, e.g., particular affinity or avidity forthe antigen.

In some embodiments, the TCR or antigen-binding portion thereof is onethat has been modified or engineered. In some embodiments, directedevolution methods are used to generate TCRs with altered properties,such as with higher affinity for a specific MHC-peptide complex. In someembodiments, directed evolution is achieved by display methodsincluding, but not limited to, yeast display (Holler et al. (2003) NatImmunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97,5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54),or T cell display (Chervin et al. (2008) J Immunol Methods, 339,175-84). In some embodiments, display approaches involve engineering, ormodifying, a known, parent or reference TCR. For example, in some cases,a wild-type TCR can be used as a template for producing mutagenized TCRsin which in one or more residues of the CDRs are mutated, and mutantswith an desired altered property, such as higher affinity for a desiredtarget antigen, are selected.

In some embodiments, peptides of a target polypeptide for use inproducing or generating a TCR of interest are known or can be readilyidentified. In some embodiments, peptides suitable for use in generatingTCRs or antigen-binding portions can be determined based on the presenceof an HLA-restricted motif in a target polypeptide of interest, such asa target polypeptide described below. In some embodiments, peptides areidentified using available computer prediction models. In someembodiments, for predicting MHC class I binding sites, such modelsinclude, but are not limited to, ProPred1 (Singh and Raghava (2001)Bioinformatics 17(12):1236-1237, and SYFPEITHI (see Schuler et al.(2007) Immunoinformatics Methods in Molecular Biology, 409(1): 75-932007). In some embodiments, the MHC-restricted epitope is HLA-A0201,which is expressed in approximately 39-46% of all Caucasians andtherefore, represents a suitable choice of MHC antigen for use preparinga TCR or other MHC-peptide binding molecule.

HLA-A0201-binding motifs and the cleavage sites for proteasomes andimmune-proteasomes using computer prediction models are known. Forpredicting MHC class I binding sites, such models include, but are notlimited to, ProPred1 (described in more detail in Singh and Raghava,ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS17(12):1236-1237 2001), and SYFPEITHI (see Schuler et al. SYFPEITHI,Database for Searching and T-Cell Epitope Prediction. inImmunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007)

In some embodiments, the TCR or antigen binding portion thereof may be arecombinantly produced natural protein or mutated form thereof in whichone or more property, such as binding characteristic, has been altered.In some embodiments, a TCR may be derived from one of various animalspecies, such as human, mouse, rat, or other mammal. A TCR may becell-bound or in soluble form. In some embodiments, for purposes of theprovided methods, the TCR is in cell-bound form expressed on the surfaceof a cell.

In some embodiments, the TCR is a full-length TCR. In some embodiments,the TCR is an antigen-binding portion. In some embodiments, the TCR is adimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR(sc-TCR). In some embodiments, a dTCR or scTCR have the structures asdescribed in WO 03/020763, WO 04/033685, WO2011/044186.

In some embodiments, the TCR contains a sequence corresponding to thetransmembrane sequence. In some embodiments, the TCR does contain asequence corresponding to cytoplasmic sequences. In some embodiments,the TCR is capable of forming a TCR complex with CD3. In someembodiments, any of the TCRs, including a dTCR or scTCR, can be linkedto signaling domains that yield an active TCR on the surface of a Tcell. In some embodiments, the TCR is expressed on the surface of cells.

In some embodiments a dTCR contains a first polypeptide wherein asequence corresponding to a TCR α chain variable region sequence isfused to the N terminus of a sequence corresponding to a TCR α chainconstant region extracellular sequence, and a second polypeptide whereina sequence corresponding to a TCR β chain variable region sequence isfused to the N terminus a sequence corresponding to a TCR β chainconstant region extracellular sequence, the first and secondpolypeptides being linked by a disulfide bond. In some embodiments, thebond can correspond to the native inter-chain disulfide bond present innative dimeric αβ TCRs. In some embodiments, the interchain disulfidebonds are not present in a native TCR. For example, in some embodiments,one or more cysteines can be incorporated into the constant regionextracellular sequences of dTCR polypeptide pair. In some cases, both anative and a non-native disulfide bond may be desirable. In someembodiments, the TCR contains a transmembrane sequence to anchor to themembrane.

In some embodiments, a dTCR contains a TCR α chain containing a variableα domain, a constant α domain and a first dimerization motif attached tothe C-terminus of the constant α domain, and a TCR β chain comprising avariable β domain, a constant β domain and a first dimerization motifattached to the C-terminus of the constant β domain, wherein the firstand second dimerization motifs easily interact to form a covalent bondbetween an amino acid in the first dimerization motif and an amino acidin the second dimerization motif linking the TCR α chain and TCR β chaintogether.

In some embodiments, the TCR is a scTCR. Typically, a scTCR can begenerated using methods known, See e.g., Soo Hoo, W. F. et al. PNAS(USA) 89, 4759 (1992); Wülfing, C. and Plückthun, A., J. Mol. Biol. 242,655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); Internationalpublished PCT Nos. WO 96/13593, WO 96/18105, WO99/60120, WO99/18129, WO03/020763, WO2011/044186; and Schlueter, C. J. et al. J. Mol. Biol. 256,859 (1996). In some embodiments, a scTCR contains an introducednon-native disulfide interchain bond to facilitate the association ofthe TCR chains (see e.g. International published PCT No. WO 03/020763).In some embodiments, a scTCR is a non-disulfide linked truncated TCR inwhich heterologous leucine zippers fused to the C-termini thereoffacilitate chain association (see e.g. International published PCT No.WO99/60120). In some embodiments, a scTCR contain a TCRα variable domaincovalently linked to a TCRβ variable domain via a peptide linker (seee.g., International published PCT No. WO99/18129).

In some embodiments, a scTCR contains a first segment constituted by anamino acid sequence corresponding to a TCR α chain variable region, asecond segment constituted by an amino acid sequence corresponding to aTCR β chain variable region sequence fused to the N terminus of an aminoacid sequence corresponding to a TCR β chain constant domainextracellular sequence, and a linker sequence linking the C terminus ofthe first segment to the N terminus of the second segment.

In some embodiments, a scTCR contains a first segment constituted by anα chain variable region sequence fused to the N terminus of an α chainextracellular constant domain sequence, and a second segment constitutedby a β chain variable region sequence fused to the N terminus of asequence β chain extracellular constant and transmembrane sequence, and,optionally, a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment.

In some embodiments, a scTCR contains a first segment constituted by aTCR β chain variable region sequence fused to the N terminus of a βchain extracellular constant domain sequence, and a second segmentconstituted by an α chain variable region sequence fused to the Nterminus of a sequence α chain extracellular constant and transmembranesequence, and, optionally, a linker sequence linking the C terminus ofthe first segment to the N terminus of the second segment.

In some embodiments, the linker of a scTCRs that links the first andsecond TCR segments can be any linker capable of forming a singlepolypeptide strand, while retaining TCR binding specificity. In someembodiments, the linker sequence may, for example, have the formula-P-AA-P- wherein P is proline and AA represents an amino acid sequencewherein the amino acids are glycine and serine. In some embodiments, thefirst and second segments are paired so that the variable regionsequences thereof are orientated for such binding. Hence, in some cases,the linker has a sufficient length to span the distance between the Cterminus of the first segment and the N terminus of the second segment,or vice versa, but is not too long to block or reduces bonding of thescTCR to the target ligand. In some embodiments, the linker can containfrom or from about 10 to 45 amino acids, such as 10 to 30 amino acids or26 to 41 amino acids residues, for example 29, 30, 31 or 32 amino acids.In some embodiments, the linker has the formula -PGGG-(SGGGG)₅-P-wherein P is proline, G is glycine and S is serine (SEQ ID NO:28). Insome embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ IDNO:29)

In some embodiments, the scTCR contains a covalent disulfide bondlinking a residue of the immunoglobulin region of the constant domain ofthe α chain to a residue of the immunoglobulin region of the constantdomain of the β chain. In some embodiments, the interchain disulfidebond in a native TCR is not present. For example, in some embodiments,one or more cysteines can be incorporated into the constant regionextracellular sequences of the first and second segments of the scTCRpolypeptide. In some cases, both a native and a non-native disulfidebond may be desirable.

In some embodiments of a dTCR or scTCR containing introduced interchaindisulfide bonds, the native disulfide bonds are not present. In someembodiments, the one or more of the native cysteines forming a nativeinterchain disulfide bonds are substituted to another residue, such asto a serine or alanine. In some embodiments, an introduced disulfidebond can be formed by mutating non-cysteine residues on the first andsecond segments to cysteine. Exemplary non-native disulfide bonds of aTCR are described in published International PCT No. WO2006/000830.

In some embodiments, the TCR or antigen-binding fragment thereofexhibits an affinity with an equilibrium binding constant for a targetantigen of between or between about 10-5 and 10-12 M and all individualvalues and ranges therein. In some embodiments, the target antigen is anMHC-peptide complex or ligand.

In some embodiments, nucleic acid or nucleic acids encoding a TCR, suchas α and β chains, can be amplified by PCR, cloning or other suitablemeans and cloned into a suitable expression vector or vectors. Theexpression vector can be any suitable recombinant expression vector, andcan be used to transform or transfect any suitable host. Suitablevectors include those designed for propagation and expansion or forexpression or both, such as plasmids and viruses.

In some embodiments, the vector can a vector of the pUC series(Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla,Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series(Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, PaloAlto, Calif.). In some cases, bacteriophage vectors, such as λG10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used. Insome embodiments, plant expression vectors can be used and includepBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In someembodiments, animal expression vectors include pEUK-Cl, pMAM and pMAMneo(Clontech). In some embodiments, a viral vector is used, such as aretroviral vector.

In some embodiments, the recombinant expression vectors can be preparedusing standard recombinant DNA techniques. In some embodiments, vectorscan contain regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost (e.g., bacterium, fungus, plant, or animal) into which the vectoris to be introduced, as appropriate and taking into considerationwhether the vector is DNA- or RNA-based. In some embodiments, the vectorcan contain a nonnative promoter operably linked to the nucleotidesequence encoding the TCR or antigen-binding portion (or otherWIC-peptide binding molecule). In some embodiments, the promoter can bea non-viral promoter or a viral promoter, such as a cytomegalovirus(CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter foundin the long-terminal repeat of the murine stem cell virus. Other knownpromoters also are contemplated.

In some embodiments, to generate a vector encoding a TCR, the α and β0chains are PCR amplified from total cDNA isolated from a T cell cloneexpressing the TCR of interest and cloned into an expression vector. Insome embodiments, the α and β chains are cloned into the same vector. Insome embodiments, the α and β chains are cloned into different vectors.In some embodiments, the generated α and β chains are incorporated intoa retroviral, e.g. lentiviral, vector. Genetically Engineered Cells andMethods of Producing Cells

In some embodiments, the provided methods involve administering to asubject having a disease or condition cells expressing a recombinantantigen receptor. Various methods for the introduction of geneticallyengineered components, e.g., recombinant receptors, e.g., CARs or TCRs,are well known and may be used with the provided methods andcompositions. Exemplary methods include those for transfer of nucleicacids encoding the receptors, including via viral, e.g., retroviral orlentiviral, transduction, transposons, and electroporation.

Among the cells expressing the receptors and administered by theprovided methods are engineered cells. The genetic engineering generallyinvolves introduction of a nucleic acid encoding the recombinant orengineered component into a composition containing the cells, such as byretroviral transduction, transfection, or transformation.

C. Chimeric Auto-Antibody Receptors (CAARs)

In some embodiments, among the recombinant receptor expressed by theengineered cells used in connection with the provided methods, uses,articles of manufacture and compositions is a chimeric autoantibodyreceptor (CAAR). In some embodiments, the CAAR is specific for anautoantibody. In some embodiments, a cell expressing the CAAR, such as aT cell engineered to express a CAAR, can be used to specifically bind toand kill autoantibody-expressing cells, but not normal antibodyexpressing cells. In some embodiments, CAAR-expressing cells can be usedto treat an autoimmune disease associated with expression ofself-antigens, such as autoimmune diseases. In some embodiments,CAAR-expressing cells can target B cells that ultimately produce theautoantibodies and display the autoantibodies on their cell surfaces,mark these B cells as disease-specific targets for therapeuticintervention. In some embodiments, CAAR-expressing cells can be used toefficiently targeting and killing the pathogenic B cells in autoimmunediseases by targeting the disease-causing B cells using anantigen-specific chimeric autoantibody receptor. In some embodiments,the recombinant receptor is a CAAR, such as any described in U.S. PatentApplication Pub. No. US 2017/0051035.

In some embodiments, the CAAR comprises an autoantibody binding domain,a transmembrane domain, and an intracellular signaling region. In someembodiments, the intracellular signaling region comprises anintracellular signaling domain. In some embodiments, the intracellularsignaling domain is or comprises a primary signaling domain, a signalingdomain that is capable of inducing a primary activation signal in a Tcell, a signaling domain of a T cell receptor (TCR) component, and/or asignaling domain comprising an immunoreceptor tyrosine-based activationmotif (ITAM). In some embodiments, the intracellular signaling regioncomprises a secondary or costimulatory signaling region (secondaryintracellular signaling regions).

In some embodiments, the autoantibody binding domain comprises anautoantigen or a fragment thereof. The choice of autoantigen can dependupon the type of autoantibody being targeted. For example, theautoantigen may be chosen because it recognizes an autoantibody on atarget cell, such as a B cell, associated with a particular diseasestate, e.g. an autoimmune disease, such as an autoantibody-mediatedautoimmune disease. In some embodiments, the autoimmune disease includespemphigus vulgaris (PV). Exemplary autoantigens include desmoglein 1(Dsg1) and Dsg3.

D. Multi-Targeting

In some embodiments, the cells used in connection with the providedmethods, uses, articles of manufacture and compositions include cellsemploying multi-targeting strategies, such as expression of two or moregenetically engineered receptors on the cell, each recognizing the sameof a different antigen and typically each including a differentintracellular signaling component. Such multi-targeting strategies aredescribed, for example, in International Patent Application, PublicationNo.: WO 2014055668 A1 (describing combinations of activating andcostimulatory CARs, e.g., targeting two different antigens presentindividually on off-target, e.g., normal cells, but present togetheronly on cells of the disease or condition to be treated) and Fedorov etal., Sci. Transl. Medicine, 5(215) (2013) (describing cells expressingan activating and an inhibitory CAR, such as those in which theactivating CAR binds to one antigen expressed on both normal ornon-diseased cells and cells of the disease or condition to be treated,and the inhibitory CAR binds to another antigen expressed only on thenormal cells or cells which it is not desired to treat).

For example, in some embodiments, the cells include a receptorexpressing a first genetically engineered antigen receptor (e.g., CAR orTCR) which is capable of inducing an activating or stimulatory signal tothe cell, generally upon specific binding to the antigen recognized bythe first receptor, e.g., the first antigen. In some embodiments, thecell further includes a second genetically engineered antigen receptor(e.g., CAR or TCR), e.g., a chimeric costimulatory receptor, which iscapable of inducing a costimulatory signal to the immune cell, generallyupon specific binding to a second antigen recognized by the secondreceptor. In some embodiments, the first antigen and second antigen arethe same. In some embodiments, the first antigen and second antigen aredifferent.

In some embodiments, the first and/or second genetically engineeredantigen receptor (e.g. CAR or TCR) is capable of inducing an activatingsignal to the cell. In some embodiments, the receptor includes anintracellular signaling component containing ITAM or ITAM-like motifs.In some embodiments, the activation induced by the first receptorinvolves a signal transduction or change in protein expression in thecell resulting in initiation of an immune response, such as ITAMphosphorylation and/or initiation of ITAM-mediated signal transductioncascade, formation of an immunological synapse and/or clustering ofmolecules near the bound receptor (e.g. CD4 or CD8, etc.), activation ofone or more transcription factors, such as NF-κB and/or AP-1, and/orinduction of gene expression of factors such as cytokines,proliferation, and/or survival.

In some embodiments, the first and/or second receptor includesintracellular signaling domains or regions of costimulatory receptorssuch as CD28, CD137 (4-1BB), OX40, and/or ICOS. In some embodiments, thefirst and second receptor include an intracellular signaling domain of acostimulatory receptor that are different. In one embodiment, the firstreceptor contains a CD28 costimulatory signaling region and the secondreceptor contain a 4-1BB co-stimulatory signaling region or vice versa.

In some embodiments, the first and/or second receptor includes both anintracellular signaling domain containing ITAM or ITAM-like motifs andan intracellular signaling domain of a costimulatory receptor.

In some embodiments, the first receptor contains an intracellularsignaling domain containing ITAM or ITAM-like motifs and the secondreceptor contains an intracellular signaling domain of a costimulatoryreceptor. The costimulatory signal in combination with the activatingsignal induced in the same cell is one that results in an immuneresponse, such as a robust and sustained immune response, such asincreased gene expression, secretion of cytokines and other factors, andT cell mediated effector functions such as cell killing.

In some embodiments, neither ligation of the first receptor alone norligation of the second receptor alone induces a robust immune response.In some aspects, if only one receptor is ligated, the cell becomestolerized or unresponsive to antigen, or inhibited, and/or is notinduced to proliferate or secrete factors or carry out effectorfunctions. In some such embodiments, however, when the plurality ofreceptors are ligated, such as upon encounter of a cell expressing thefirst and second antigens, a desired response is achieved, such as fullimmune activation or stimulation, e.g., as indicated by secretion of oneor more cytokine, proliferation, persistence, and/or carrying out animmune effector function such as cytotoxic killing of a target cell.

In some embodiments, the two receptors induce, respectively, anactivating and an inhibitory signal to the cell, such that binding byone of the receptor to its antigen activates the cell or induces aresponse, but binding by the second inhibitory receptor to its antigeninduces a signal that suppresses or dampens that response. Examples arecombinations of activating CARs and inhibitory CARs or iCARs. Such astrategy may be used, for example, in which the activating CAR binds anantigen expressed in a disease or condition but which is also expressedon normal cells, and the inhibitory receptor binds to a separate antigenwhich is expressed on the normal cells but not cells of the disease orcondition.

In some embodiments, the multi-targeting strategy is employed in a casewhere an antigen associated with a particular disease or condition isexpressed on a non-diseased cell and/or is expressed on the engineeredcell itself, either transiently (e.g., upon stimulation in associationwith genetic engineering) or permanently. In such cases, by requiringligation of two separate and individually specific antigen receptors,specificity, selectivity, and/or efficacy may be improved.

In some embodiments, the plurality of antigens, e.g., the first andsecond antigens, are expressed on the cell, tissue, or disease orcondition being targeted, such as on the cancer cell. In some aspects,the cell, tissue, disease or condition is multiple myeloma or a multiplemyeloma cell. In some embodiments, one or more of the plurality ofantigens generally also is expressed on a cell which it is not desiredto target with the cell therapy, such as a normal or non-diseased cellor tissue, and/or the engineered cells themselves. In such embodiments,by requiring ligation of multiple receptors to achieve a response of thecell, specificity and/or efficacy is achieved.

E. Vectors and Methods for Genetic Engineering

In some embodiments, engineered cells, such as T cells, used inconnection with the provided methods, uses, articles of manufacture orcompositions are cells have been genetically engineered to express arecombinant receptor, e.g., a CAR or a TCR described herein. In someembodiments, the cells are engineered by introduction, delivery ortransfer of nucleic acid sequences that encode the recombinant receptorand/or other molecules.

In some embodiments, recombinant nucleic acids are transferred intocells using recombinant infectious virus particles, such as, e.g.,vectors derived from simian virus 40 (SV40), adenoviruses,adeno-associated virus (AAV). In some embodiments, recombinant nucleicacids are transferred into T cells using recombinant lentiviral vectorsor retroviral vectors, such as gamma-retroviral vectors (see, e.g.,Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi: 10.1038/gt.2014.25;Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al.(2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011Nov. 29 (11): 550-557.

In some embodiments, the retroviral vector has a long terminal repeatsequence (LTR), e.g., a retroviral vector derived from the Moloneymurine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV),murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV),spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Mostretroviral vectors are derived from murine retroviruses. In someembodiments, the retroviruses include those derived from any avian ormammalian cell source. The retroviruses typically are amphotropic,meaning that they are capable of infecting host cells of severalspecies, including humans. In one embodiment, the gene to be expressedreplaces the retroviral gag, pol and/or env sequences. A number ofillustrative retroviral systems have been described (e.g., U.S. Pat.Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993)Cur. Opin. Genet. Develop. 3:102-109.

Methods of lentiviral transduction are known. Exemplary methods aredescribed in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701;Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009)Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood.102(2): 497-505.

In some embodiments, recombinant nucleic acids are transferred into Tcells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids aretransferred into T cells via transposition (see, e.g., Manuri et al.(2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec TherNucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic materialin immune cells include calcium phosphate transfection (e.g., asdescribed in Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.), protoplast fusion, cationic liposome-mediatedtransfection; tungsten particle-facilitated microparticle bombardment(Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNAco-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transfer of the nucleic acids encodingthe recombinant products are those described, e.g., in internationalpatent application, Publication No.: WO2014055668, and U.S. Pat. No.7,446,190.

In some embodiments, the cells, e.g., T cells, may be transfected eitherduring or after expansion e.g. with a T cell receptor (TCR) or achimeric antigen receptor (CAR). This transfection for the introductionof the gene of the desired receptor can be carried out with any suitableretroviral vector, for example. The genetically modified cell populationcan then be liberated from the initial stimulus (the anti-CD3/anti-CD28stimulus, for example) and subsequently be stimulated with a second typeof stimulus e.g. via a de novo introduced receptor). This second type ofstimulus may include an antigenic stimulus in form of a peptide/MHCmolecule, the cognate (cross-linking) ligand of the geneticallyintroduced receptor (e.g. natural ligand of a CAR) or any ligand (suchas an antibody) that directly binds within the framework of the newreceptor (e.g. by recognizing constant regions within the receptor).See, for example, Cheadle et al, “Chimeric antigen receptors for T-cellbased therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al.,Chimeric Antigen Receptor Therapy for Cancer Annual Review of MedicineVol. 65: 333-347 (2014).

In some cases, a vector may be used that does not require that thecells, e.g., T cells, are activated. In some such instances, the cellsmay be selected and/or transduced prior to activation. Thus, the cellsmay be engineered prior to, or subsequent to culturing of the cells, andin some cases at the same time as or during at least a portion of theculturing.

Among additional nucleic acids, e.g., genes for introduction are thoseto improve the efficacy of therapy, such as by promoting viabilityand/or function of transferred cells; genes to provide a genetic markerfor selection and/or evaluation of the cells, such as to assess in vivosurvival or localization; genes to improve safety, for example, bymaking the cell susceptible to negative selection in vivo as describedby Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell etal., Human Gene Therapy 3:319-338 (1992); see also the publications ofPCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use ofbifunctional selectable fusion genes derived from fusing a dominantpositive selectable marker with a negative selectable marker. See, e.g.,Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.

F. Cells and Preparation of Cells for Genetic Engineering

In some embodiments, cells, such as T cells, used in connection with theprovided methods, uses, articles of manufacture or compositions arecells have been genetically engineered to express a recombinantreceptor, e.g., a CAR or a TCR described herein. In some embodiments,the engineered cells are used in the context of cell therapy, e.g.,adoptive cell therapy. In some embodiments, the engineered cells areimmune cells. In some embodiments, the engineered cells are T cells,such as CD4+ or CD8+ T cells.

In some embodiments, the nucleic acids, such as nucleic acids encoding arecombinant receptor, are heterologous, i.e., normally not present in acell or sample obtained from the cell, such as one obtained from anotherorganism or cell, which for example, is not ordinarily found in the cellbeing engineered and/or an organism from which such cell is derived. Insome embodiments, the nucleic acids are not naturally occurring, such asa nucleic acid not found in nature, including one comprising chimericcombinations of nucleic acids encoding various domains from multipledifferent cell types.

The cells generally are eukaryotic cells, such as mammalian cells, andtypically are human cells. In some embodiments, the cells are derivedfrom the blood, bone marrow, lymph, or lymphoid organs, are cells of theimmune system, such as cells of the innate or adaptive immunity, e.g.,myeloid or lymphoid cells, including lymphocytes, typically T cellsand/or NK cells. Other exemplary cells include stem cells, such asmultipotent and pluripotent stem cells, including induced pluripotentstem cells (iPSCs). The cells typically are primary cells, such as thoseisolated directly from a subject and/or isolated from a subject andfrozen. In some embodiments, the cells include one or more subsets of Tcells or other cell types, such as whole T cell populations, CD4⁺ cells,CD8⁺ cells, and subpopulations thereof, such as those defined byfunction, activation state, maturity, potential for differentiation,expansion, recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. With reference to the subject to be treated,the cells may be allogeneic and/or autologous. Among the methods includeoff-the-shelf methods. In some aspects, such as for off-the-shelftechnologies, the cells are pluripotent and/or multipotent, such as stemcells, such as induced pluripotent stem cells (iPSCs). In someembodiments, the methods include isolating cells from the subject,preparing, processing, culturing, and/or engineering them, andre-introducing them into the same subject, before or aftercryopreservation.

Among the sub-types and subpopulations of T cells and/or of CD4⁺ and/orof CD8⁺ T cells are naïve T (T_(N)) cells, effector T cells (T_(EFF)),memory T cells and sub-types thereof, such as stem cell memory T(T_(SCM)), central memory T (T_(CM)), effector memory T (T_(EM)), orterminally differentiated effector memory T cells, tumor-infiltratinglymphocytes (TIL), immature T cells, mature T cells, helper T cells,cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturallyoccurring and adaptive regulatory T (Treg) cells, helper T cells, suchas TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells,follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, the cells are natural killer (NK) cells. In someembodiments, the cells are monocytes or granulocytes, e.g., myeloidcells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils.

In some embodiments, the cells include one or more nucleic acidsintroduced via genetic engineering, and thereby express recombinant orgenetically engineered products of such nucleic acids. In someembodiments, the nucleic acids are heterologous, i.e., normally notpresent in a cell or sample obtained from the cell, such as one obtainedfrom another organism or cell, which for example, is not ordinarilyfound in the cell being engineered and/or an organism from which suchcell is derived. In some embodiments, the nucleic acids are notnaturally occurring, such as a nucleic acid not found in nature,including one comprising chimeric combinations of nucleic acids encodingvarious domains from multiple different cell types.

In some embodiments, preparation of the engineered cells includes one ormore culture and/or preparation steps. The cells for introduction of thenucleic acid encoding the transgenic receptor such as the CAR, may beisolated from a sample, such as a biological sample, e.g., one obtainedfrom or derived from a subject. In some embodiments, the subject fromwhich the cell is isolated is one having the disease or condition or inneed of a cell therapy or to which cell therapy will be administered.The subject in some embodiments is a human in need of a particulartherapeutic intervention, such as the adoptive cell therapy for whichcells are being isolated, processed, and/or engineered.

Accordingly, the cells in some embodiments are primary cells, e.g.,primary human cells. The samples include tissue, fluid, and othersamples taken directly from the subject, as well as samples resultingfrom one or more processing steps, such as separation, centrifugation,genetic engineering (e.g. transduction with viral vector), washing,and/or incubation. The biological sample can be a sample obtaineddirectly from a biological source or a sample that is processed.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples, including processed samples derivedtherefrom.

In some aspects, the sample from which the cells are derived or isolatedis blood or a blood-derived sample, or is or is derived from anapheresis or leukapheresis product. Exemplary samples include wholeblood, peripheral blood mononuclear cells (PBMCs), leukocytes, bonemarrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node,gut associated lymphoid tissue, mucosa associated lymphoid tissue,spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon,kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries,tonsil, or other organ, and/or cells derived therefrom. Samples include,in the context of cell therapy, e.g., adoptive cell therapy, samplesfrom autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells in some embodiments are obtained from a xenogeneicsource, for example, from mouse, rat, non-human primate, and pig.

In some embodiments, isolation of the cells includes one or morepreparation and/or non-affinity based cell separation steps. In someexamples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someaspects, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in some aspects contains cells other thanred blood cells and platelets.

In some embodiments, the blood cells collected from the subject arewashed, e.g., to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In someembodiments, the cells are washed with phosphate buffered saline (PBS).In some embodiments, the wash solution lacks calcium and/or magnesiumand/or many or all divalent cations. In some aspects, a washing step isaccomplished a semi-automated “flow-through” centrifuge (for example,the Cobe 2991 cell processor, Baxter) according to the manufacturer'sinstructions. In some aspects, a washing step is accomplished bytangential flow filtration (TFF) according to the manufacturer'sinstructions. In some embodiments, the cells are resuspended in avariety of biocompatible buffers after washing, such as, for example,Ca⁺⁺/Mg⁺⁺ free PBS. In certain embodiments, components of a blood cellsample are removed and the cells directly resuspended in culture media.

In some embodiments, the methods include density-based cell separationmethods, such as the preparation of white blood cells from peripheralblood by lysing the red blood cells and centrifugation through a Percollor Ficoll gradient.

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In some examples, both fractions areretained for further use. In some aspects, negative selection can beparticularly useful where no antibody is available that specificallyidentifies a cell type in a heterogeneous population, such thatseparation is best carried out based on markers expressed by cells otherthan the desired population.

The separation need not result in 100% enrichment or removal of aparticular cell population or cells expressing a particular marker. Forexample, positive selection of or enrichment for cells of a particulartype, such as those expressing a marker, refers to increasing the numberor percentage of such cells, but need not result in a complete absenceof cells not expressing the marker. Likewise, negative selection,removal, or depletion of cells of a particular type, such as thoseexpressing a marker, refers to decreasing the number or percentage ofsuch cells, but need not result in a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

For example, in some aspects, specific subpopulations of T cells, suchas cells positive or expressing high levels of one or more surfacemarkers, e.g., CD28⁺, CD62L⁺, CCR7⁺, CD27⁺, CD127⁺, CD4⁺, CD8⁺, CD45RA⁺,and/or CD45RO⁺ T cells, are isolated by positive or negative selectiontechniques.

For example, CD3⁺, CD28⁺ T cells can be positively selected usinganti-CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450CD3/CD28 T Cell Expander).

In some embodiments, isolation is carried out by enrichment for aparticular cell population by positive selection, or depletion of aparticular cell population, by negative selection. In some embodiments,positive or negative selection is accomplished by incubating cells withone or more antibodies or other binding agent that specifically bind toone or more surface markers expressed or expressed (marker⁺) at arelatively higher level (marker^(high)) on the positively or negativelyselected cells, respectively.

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In some aspects, aCD4⁺ or CD8⁺ selection step is used to separate CD4⁺ helper and CD8⁺cytotoxic T cells. Such CD4⁺ and CD8⁺ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations.

In some embodiments, CD8⁺ cells are further enriched for or depleted ofnaive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In someembodiments, enrichment for central memory T (T^(CM)) cells is carriedout to increase efficacy, such as to improve long-term survival,expansion, and/or engraftment following administration, which in someaspects is particularly robust in such sub-populations. See Terakura etal. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother.35(9):689-701. In some embodiments, combining T_(CM)-enriched CD8⁺ Tcells and CD4⁺ T cells further enhances efficacy.

In embodiments, memory T cells are present in both CD62L⁺ and CD62L⁻subsets of CD8⁺ peripheral blood lymphocytes. PBMC can be enriched foror depleted of CD62L⁻CD8⁺ and/or CD62L⁺CD8⁺ fractions, such as usinganti-CD8 and anti-CD62L antibodies.

In some embodiments, the enrichment for central memory T (T_(CM)) cellsis based on positive or high surface expression of CD45RO, CD62L, CCR7,CD28, CD3, and/or CD127; in some aspects, it is based on negativeselection for cells expressing or highly expressing CD45RA and/orgranzyme B. In some aspects, isolation of a CD8⁺ population enriched forT_(CM) cells is carried out by depletion of cells expressing CD4, CD14,CD45RA, and positive selection or enrichment for cells expressing CD62L.In one aspect, enrichment for central memory T (T_(CM)) cells is carriedout starting with a negative fraction of cells selected based on CD4expression, which is subjected to a negative selection based onexpression of CD14 and CD45RA, and a positive selection based on CD62L.Such selections in some aspects are carried out simultaneously and inother aspects are carried out sequentially, in either order. In someaspects, the same CD4 expression-based selection step used in preparingthe CD8⁺ cell population or subpopulation, also is used to generate theCD4⁺ cell population or subpopulation, such that both the positive andnegative fractions from the CD4-based separation are retained and usedin subsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

In a particular example, a sample of PBMCs or other white blood cellsample is subjected to selection of CD4⁺ cells, where both the negativeand positive fractions are retained. The negative fraction then issubjected to negative selection based on expression of CD14 and CD45RAor CD19, and positive selection based on a marker characteristic ofcentral memory T cells, such as CD62L or CCR7, where the positive andnegative selections are carried out in either order.

CD4⁺ T helper cells are sorted into naïve, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4⁺ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4⁺ T lymphocytes are CD45RO⁻, CD45RA⁺, CD62L⁺, CD4⁺T cells. In some embodiments, central memory CD4⁺ cells are CD62L⁺ andCD45RO⁺. In some embodiments, effector CD4⁺ cells are CD62L⁻ andCD45RO⁻.

In one example, to enrich for CD4⁺ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody orbinding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection. For example, in some embodiments,the cells and cell populations are separated or isolated usingimmunomagnetic (or affinity magnetic) separation techniques (reviewed inMethods in Molecular Medicine, vol. 58: Metastasis Research Protocols,Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A.Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).

In some aspects, the sample or composition of cells to be separated isincubated with small, magnetizable or magnetically responsive material,such as magnetically responsive particles or microparticles, such asparamagnetic beads (e.g., such as Dynalbeads or MACS beads). Themagnetically responsive material, e.g., particle, generally is directlyor indirectly attached to a binding partner, e.g., an antibody, thatspecifically binds to a molecule, e.g., surface marker, present on thecell, cells, or population of cells that it is desired to separate,e.g., that it is desired to negatively or positively select.

In some embodiments, the magnetic particle or bead comprises amagnetically responsive material bound to a specific binding member,such as an antibody or other binding partner. There are many well-knownmagnetically responsive materials used in magnetic separation methods.Suitable magnetic particles include those described in Molday, U.S. Pat.No. 4,452,773, and in European Patent Specification EP 452342 B, whichare hereby incorporated by reference. Colloidal sized particles, such asthose described in Owen U.S. Pat. No. 4,795,698, and Liberti et al.,U.S. Pat. No. 5,200,084 are other examples.

The incubation generally is carried out under conditions whereby theantibodies or binding partners, or molecules, such as secondaryantibodies or other reagents, which specifically bind to such antibodiesor binding partners, which are attached to the magnetic particle orbead, specifically bind to cell surface molecules if present on cellswithin the sample.

In some aspects, the sample is placed in a magnetic field, and thosecells having magnetically responsive or magnetizable particles attachedthereto will be attracted to the magnet and separated from the unlabeledcells. For positive selection, cells that are attracted to the magnetare retained; for negative selection, cells that are not attracted(unlabeled cells) are retained. In some aspects, a combination ofpositive and negative selection is performed during the same selectionstep, where the positive and negative fractions are retained and furtherprocessed or subject to further separation steps.

In certain embodiments, the magnetically responsive particles are coatedin primary antibodies or other binding partners, secondary antibodies,lectins, enzymes, or streptavidin. In certain embodiments, the magneticparticles are attached to cells via a coating of primary antibodiesspecific for one or more markers. In certain embodiments, the cells,rather than the beads, are labeled with a primary antibody or bindingpartner, and then cell-type specific secondary antibody- or otherbinding partner (e.g., streptavidin)-coated magnetic particles, areadded. In certain embodiments, streptavidin-coated magnetic particlesare used in conjunction with biotinylated primary or secondaryantibodies.

In some embodiments, the magnetically responsive particles are leftattached to the cells that are to be subsequently incubated, culturedand/or engineered; in some aspects, the particles are left attached tothe cells for administration to a patient. In some embodiments, themagnetizable or magnetically responsive particles are removed from thecells. Methods for removing magnetizable particles from cells are knownand include, e.g., the use of competing non-labeled antibodies, andmagnetizable particles or antibodies conjugated to cleavable linkers. Insome embodiments, the magnetizable particles are biodegradable.

In some embodiments, the affinity-based selection is viamagnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn,Calif.). Magnetic Activated Cell Sorting (MACS) systems are capable ofhigh-purity selection of cells having magnetized particles attachedthereto. In certain embodiments, MACS operates in a mode wherein thenon-target and target species are sequentially eluted after theapplication of the external magnetic field. That is, the cells attachedto magnetized particles are held in place while the unattached speciesare eluted. Then, after this first elution step is completed, thespecies that were trapped in the magnetic field and were prevented frombeing eluted are freed in some manner such that they can be eluted andrecovered. In certain embodiments, the non-target cells are labelled anddepleted from the heterogeneous population of cells.

In certain embodiments, the isolation or separation is carried out usinga system, device, or apparatus that carries out one or more of theisolation, cell preparation, separation, processing, incubation,culture, and/or formulation steps of the methods. In some aspects, thesystem is used to carry out each of these steps in a closed or sterileenvironment, for example, to minimize error, user handling and/orcontamination. In one example, the system is a system as described inInternational Patent Application, Publication Number WO2009/072003, orUS 20110003380 A1.

In some embodiments, the system or apparatus carries out one or more,e.g., all, of the isolation, processing, engineering, and formulationsteps in an integrated or self-contained system, and/or in an automatedor programmable fashion. In some aspects, the system or apparatusincludes a computer and/or computer program in communication with thesystem or apparatus, which allows a user to program, control, assess theoutcome of, and/or adjust various aspects of the processing, isolation,engineering, and formulation steps.

In some aspects, the separation and/or other steps is carried out usingCliniMACS system (Miltenyi Biotec), for example, for automatedseparation of cells on a clinical-scale level in a closed and sterilesystem. Components can include an integrated microcomputer, magneticseparation unit, peristaltic pump, and various pinch valves. Theintegrated computer in some aspects controls all components of theinstrument and directs the system to perform repeated procedures in astandardized sequence. The magnetic separation unit in some aspectsincludes a movable permanent magnet and a holder for the selectioncolumn. The peristaltic pump controls the flow rate throughout thetubing set and, together with the pinch valves, ensures the controlledflow of buffer through the system and continual suspension of cells.

The CliniMACS system in some aspects uses antibody-coupled magnetizableparticles that are supplied in a sterile, non-pyrogenic solution. Insome embodiments, after labelling of cells with magnetic particles thecells are washed to remove excess particles. A cell preparation bag isthen connected to the tubing set, which in turn is connected to a bagcontaining buffer and a cell collection bag. The tubing set consists ofpre-assembled sterile tubing, including a pre-column and a separationcolumn, and are for single use only. After initiation of the separationprogram, the system automatically applies the cell sample onto theseparation column. Labelled cells are retained within the column, whileunlabeled cells are removed by a series of washing steps. In someembodiments, the cell populations for use with the methods describedherein are unlabeled and are not retained in the column. In someembodiments, the cell populations for use with the methods describedherein are labeled and are retained in the column. In some embodiments,the cell populations for use with the methods described herein areeluted from the column after removal of the magnetic field, and arecollected within the cell collection bag.

In certain embodiments, separation and/or other steps are carried outusing the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACSProdigy system in some aspects is equipped with a cell processing unitythat permits automated washing and fractionation of cells bycentrifugation. The CliniMACS Prodigy system can also include an onboardcamera and image recognition software that determines the optimal cellfractionation endpoint by discerning the macroscopic layers of thesource cell product. For example, peripheral blood is automaticallyseparated into erythrocytes, white blood cells and plasma layers. TheCliniMACS Prodigy system can also include an integrated cell cultivationchamber which accomplishes cell culture protocols such as, e.g., celldifferentiation and expansion, antigen loading, and long-term cellculture. Input ports can allow for the sterile removal and replenishmentof media and cells can be monitored using an integrated microscope. See,e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura etal. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother.35(9):689-701.

In some embodiments, a cell population described herein is collected andenriched (or depleted) via flow cytometry, in which cells stained formultiple cell surface markers are carried in a fluidic stream. In someembodiments, a cell population described herein is collected andenriched (or depleted) via preparative scale (FACS)-sorting. In certainembodiments, a cell population described herein is collected andenriched (or depleted) by use of microelectromechanical systems (MEMS)chips in combination with a FACS-based detection system (see, e.g., WO2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al.(2008) J Biophoton. 1(5):355-376. In both cases, cells can be labeledwith multiple markers, allowing for the isolation of well-defined T cellsubsets at high purity.

In some embodiments, the antibodies or binding partners are labeled withone or more detectable marker, to facilitate separation for positiveand/or negative selection. For example, separation may be based onbinding to fluorescently labeled antibodies. In some examples,separation of cells based on binding of antibodies or other bindingpartners specific for one or more cell surface markers are carried in afluidic stream, such as by fluorescence-activated cell sorting (FACS),including preparative scale (FACS) and/or microelectromechanical systems(MEMS) chips, e.g., in combination with a flow-cytometric detectionsystem. Such methods allow for positive and negative selection based onmultiple markers simultaneously.

In some embodiments, the preparation methods include steps for freezing,e.g., cryopreserving, the cells, either before or after isolation,incubation, and/or engineering. In some embodiments, the freeze andsubsequent thaw step removes granulocytes and, to some extent, monocytesin the cell population. In some embodiments, the cells are suspended ina freezing solution, e.g., following a washing step to remove plasma andplatelets. Any of a variety of known freezing solutions and parametersin some aspects may be used. One example involves using PBS containing20% DMSO and 8% human serum albumin (HSA), or other suitable cellfreezing media. This is then diluted 1:1 with media so that the finalconcentration of DMSO and HSA are 10% and 4%, respectively. The cellsare generally then frozen to −80° C. at a rate of 1° per minute andstored in the vapor phase of a liquid nitrogen storage tank.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Theincubation and/or engineering may be carried out in a culture vessel,such as a unit, chamber, well, column, tube, tubing set, valve, vial,culture dish, bag, or other container for culture or cultivating cells.In some embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor.

The conditions can include one or more of particular media, temperature,oxygen content, carbon dioxide content, time, agents, e.g., nutrients,amino acids, antibiotics, ions, and/or stimulatory factors, such ascytokines, chemokines, antigens, binding partners, fusion proteins,recombinant soluble receptors, and any other agents designed to activatethe cells.

In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT cell. Such agents can include antibodies, such as those specific for aTCR, e.g. anti-CD3. In some embodiments, the stimulating conditionsinclude one or more agent, e.g. ligand, which is capable of stimulatinga costimulatory receptor, e.g., anti-CD28. In some embodiments, suchagents and/or ligands may be, bound to solid support such as a bead,and/or one or more cytokines. Optionally, the expansion method mayfurther comprise the step of adding anti-CD3 and/or anti-CD28 antibodyto the culture medium (e.g., at a concentration of at least about 0.5ng/ml). In some embodiments, the stimulating agents include IL-2, IL-15and/or IL-7. In some aspects, the IL-2 concentration is at least about10 units/mL.

In some aspects, incubation is carried out in accordance with techniquessuch as those described in U.S. Pat. No. 6,040,177 to Riddell et al.,Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al.(2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother.35(9):689-701.

In some embodiments, the T cells are expanded by adding to aculture-initiating composition feeder cells, such as non-dividingperipheral blood mononuclear cells (PBMC), (e.g., such that theresulting population of cells contains at least about 5, 10, 20, or 40or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T cells). In some aspects, thenon-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In some embodiments, the PBMC are irradiated with gamma rays inthe range of about 3000 to 3600 rads to prevent cell division. In someaspects, the feeder cells are added to culture medium prior to theaddition of the populations of T cells.

In some embodiments, the stimulating conditions include temperaturesuitable for the growth of human T lymphocytes, for example, at leastabout 25 degrees Celsius, generally at least about 30 degrees, andgenerally at or about 37 degrees Celsius. Optionally, the incubation mayfurther comprise adding non-dividing EBV-transformed lymphoblastoidcells (LCL) as feeder cells. LCL can be irradiated with gamma rays inthe range of about 6000 to 10,000 rads. The LCL feeder cells in someaspects is provided in any suitable amount, such as a ratio of LCLfeeder cells to initial T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4⁺and/or CD8⁺ T cells, are obtained by stimulating naive or antigenspecific T lymphocytes with antigen. For example, antigen-specific Tcell lines or clones can be generated to cytomegalovirus antigens byisolating T cells from infected subjects and stimulating the cells invitro with the same antigen.

IV. Compositions and Formulations

In some embodiments, the dose of cells comprising cells engineered witha recombinant antigen receptor, e.g. CAR or TCR, is provided as acomposition or formulation, such as a pharmaceutical composition orformulation. Such compositions can be used in accord with the providedmethods or uses, and/or with the provided articles of manufacture orcompositions, such as in the prevention or treatment of diseases,conditions, and disorders, or in detection, diagnostic, and prognosticmethods.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

In some aspects, the choice of carrier is determined in part by theparticular cell or agent and/or by the method of administration.Accordingly, there are a variety of suitable formulations. For example,the pharmaceutical composition can contain preservatives. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. In some aspects, a mixtureof two or more preservatives is used. The preservative or mixturesthereof are typically present in an amount of about 0.0001% to about 2%by weight of the total composition. Carriers are described, e.g., byRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG).

Buffering agents in some aspects are included in the compositions.Suitable buffering agents include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. In some aspects, a mixture of two or more buffering agents isused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition. Methods for preparing administrable pharmaceuticalcompositions are known. Exemplary methods are described in more detailin, for example, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulation or composition may also contain more than one activeingredient useful for the particular indication, disease, or conditionbeing prevented or treated with the cells or agents, where therespective activities do not adversely affect one another. Such activeingredients are suitably present in combination in amounts that areeffective for the purpose intended. Thus, in some embodiments, thepharmaceutical composition further includes other pharmaceuticallyactive agents or drugs, such as chemotherapeutic agents, e.g.,asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, vincristine, etc. In someembodiments, the agents or cells are administered in the form of a salt,e.g., a pharmaceutically acceptable salt. Suitable pharmaceuticallyacceptable acid addition salts include those derived from mineral acids,such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric,and sulphuric acids, and organic acids, such as tartaric, acetic,citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic,and arylsulphonic acids, for example, p-toluenesulphonic acid.

The pharmaceutical composition in some embodiments contains agents orcells in amounts effective to treat or prevent the disease or condition,such as a therapeutically effective or prophylactically effectiveamount. Therapeutic or prophylactic efficacy in some embodiments ismonitored by periodic assessment of treated subjects. For repeatedadministrations over several days or longer, depending on the condition,the treatment is repeated until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful and can bedetermined. The desired dosage can be delivered by a single bolusadministration of the composition, by multiple bolus administrations ofthe composition, or by continuous infusion administration of thecomposition.

The agents or cells can be administered by any suitable means, forexample, by bolus infusion, by injection, e.g., intravenous orsubcutaneous injections, intraocular injection, periocular injection,subretinal injection, intravitreal injection, trans-septal injection,subscleral injection, intrachoroidal injection, intracameral injection,subconjectval injection, subconjuntival injection, sub-Tenon'sinjection, retrobulbar injection, peribulbar injection, or posteriorjuxtascleral delivery. In some embodiments, they are administered byparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In some embodiments, a given dose isadministered by a single bolus administration of the cells or agent. Insome embodiments, it is administered by multiple bolus administrationsof the cells or agent, for example, over a period of no more than 3days, or by continuous infusion administration of the cells or agent.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of agent oragents, the type of cells or recombinant receptors, the severity andcourse of the disease, whether the agent or cells are administered forpreventive or therapeutic purposes, previous therapy, the subject'sclinical history and response to the agent or the cells, and thediscretion of the attending physician. The compositions are in someembodiments suitably administered to the subject at one time or over aseries of treatments.

The cells or agents may be administered using standard administrationtechniques, formulations, and/or devices. Provided are formulations anddevices, such as syringes and vials, for storage and administration ofthe compositions. With respect to cells, administration can beautologous or heterologous. For example, immunoresponsive cells orprogenitors can be obtained from one subject, and administered to thesame subject or a different, compatible subject. Peripheral bloodderived immunoresponsive cells or their progeny (e.g., in vivo, ex vivoor in vitro derived) can be administered via localized injection,including catheter administration, systemic injection, localizedinjection, intravenous injection, or parenteral administration. Whenadministering a therapeutic composition (e.g., a pharmaceuticalcomposition containing a genetically modified immunoresponsive cell oran agent that treats or ameliorates symptoms of neurotoxicity), it willgenerally be formulated in a unit dosage injectable form (solution,suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In some embodiments, theagent or cell populations are administered parenterally. The term“parenteral,” as used herein, includes intravenous, intramuscular,subcutaneous, rectal, vaginal, and intraperitoneal administration. Insome embodiments, the agent or cell populations are administered to asubject using peripheral systemic delivery by intravenous,intraperitoneal, or subcutaneous injection.

Compositions in some embodiments are provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may in some aspects bebuffered to a selected pH. Liquid preparations are normally easier toprepare than gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection. Viscous compositions, on the otherhand, can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the agentor cells in a solvent, such as in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose, dextrose, or the like.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

V. Combination Therapy

In some embodiments of the methods, articles of manufacture, uses orcompositions, the cell therapy, e.g. dose of T cells (e.g. CAR⁺ T cells)is administered to subjects in combination with an additionaltherapeutic agent or therapy, generally other than the cell therapy oranother cell therapy, such as other than a CAR⁺ T cell therapy. In someembodiments, the cell therapy, e.g. dose of genetically engineered Tcells, such as CAR⁺ T cells, in the provided methods or uses, and/orwith the articles of manufacture or compositions, is administered aspart of a combination treatment or combination therapy, such assimultaneously with, sequentially with or intermittently with, in anyorder, one or more additional therapeutic intervention. In someembodiments, the one or more additional therapeutic interventionincludes any agent or treatment for treating or preventing the diseaseor condition, such as the B cell malignancy, e.g. NHL, and/or any agentor treatment to increase the efficacy, persistence, and/or activity ofthe engineered cell therapy.

In some embodiments, an additional therapeutic agent or therapy isadministered to subjects who are or are likely to be or who arepredicted to be poor responders and/or who do not, are likely not toand/or who are predicted not to respond or do not respond within acertain time and/or to a certain extent to treatment with the celltherapy, e.g. dose of T cells (e.g. CAR⁺ T cells). In some embodiments,the additional therapeutic agent is administered to subjects who do notor are not likely to or are not predicted to exhibit a complete responseor overall response, such as within 1 month, within two months or withinthree months after initiation of administration of the cell therapy. Insome embodiments, the additional therapeutic agent is administered tosubjects who exhibit or are likely to exhibit or who are predicted toexhibit progressive disease (PD), such as within 1 month, two months orthree months, following administration of the cell therapy. In someembodiments, a subject is likely or predicted not to exhibit a responseor a certain response based on a plurality of similarly situatedsubjects so treated or previously treated with the cell therapy.

In some embodiments, it is observed that a subject that may or that ismore likely to exhibit a poor response to cell therapy, e.g. dose of Tcells (e.g. CAR⁺ T cells) includes a subject with NHL that is or hasbeen identified to have stable or progressive disease (SD/PD) followingtreatment with a prior therapy, optionally a prior therapy with achemotherapeutic agent, that is or has been identified with an EasternCooperative Oncology Group Performance Status (ECOG) status of 2, thatis or has been identified as having a transformed follicular lymphoma(tFL), or that is or has been identified has having a DLBCL transformedfrom MZL and CLL. In some embodiments, the provided methods includeselecting a subject that is or is likely to exhibit a poor response to acell therapy when the cell therapy is administered alone, andadministering the cell therapy in combination with an additional agentor therapy, such as any as described. In some embodiments, the a subjectfor treatment in the provided combination therapy methods is a subjectthat is selected as having a B cell malignancy, such as NHL, and thathas stable or progressive disease (SD/PD) following treatment with aprior therapy, optionally a prior therapy with a chemotherapeutic agent,that has an Eastern Cooperative Oncology Group Performance Status (ECOG)status of 2, that has a transformed follicular lymphoma (tFL), or thathas a DLBCL transformed from MZL and CLL. In some embodiments, theadditional agent or therapy can be administered prior to, concomitantlywith or at the same time and/or subsequently to initiation ofadministration of the cell therapy, e.g. dose of T cells (e.g. CAR⁺ Tcells).

In certain embodiments, it is found that the pharmacokinetics (PK) ofthe cell therapy in the blood of subjects following administration ofthe cell therapy is similar or not substantially different betweensubjects that respond (e.g. exhibit a CR or OR) versus do not respond(e.g. exhibit PD) to the cell therapy. In some embodiments, suchobservations indicate that the cell therapy has or is expanding in thesubject but may not exhibit optimal efficacy.

In some contexts, optimal efficacy of a cell therapy can depend on theability of the administered cells to recognize and bind to a target,e.g., target antigen, to traffic, localize to and successfully enterappropriate sites within the subject, tumors, and environments thereof.In some contexts, optimal efficacy can depend on the ability of theadministered cells to become activated, expand, to exert variouseffector functions, including cytotoxic killing and secretion of variousfactors such as cytokines, to persist, including long-term, todifferentiate, transition or engage in reprogramming into certainphenotypic states (such as long-lived memory, less-differentiated, andeffector states), to avoid or reduce immunosuppressive conditions in thelocal microenvironment of a disease, to provide effective and robustrecall responses following clearance and re-exposure to target ligand orantigen, and avoid or reduce exhaustion, anergy, peripheral tolerance,terminal differentiation, and/or differentiation into a suppressivestate.

In some aspects, the efficacy of the immunotherapy, e.g., T celltherapy, may be limited by the immunosuppressive activity or factorspresent in the local microenvironment of the disease or disorder, e.g.,the TME. In some aspects, the TME contains or produces factors orconditions that can suppress the activity, function, proliferation,survival and/or persistence of T cells administered for T cell therapy.

In some embodiments, administration of an additional agent or therapy,prior to, concomitantly with or at the same time and/or subsequently toinitiation of administration of the cell therapy, e.g. dose of T cells(e.g. CAR⁺ T cells) can result in improved activity, efficacy and/orpersistence of the cell therapy and/or improve responses of the treatedsubject. In some embodiments, the additional agent for combinationtreatment or combination therapy enhances, boosts and/or promotes theefficacy and/or safety of the therapeutic effect of the cell therapy,e.g. engineered T cell therapy, such as CAR⁺ T cells. In someembodiments, the additional agent enhances or improves the efficacy,survival or persistence of the administered cells, e.g., cellsexpressing the recombinant receptor, e.g. CAR.

In some embodiments, the additional agent of therapy is an antibody or acytotoxic or therapeutic agent, e.g., a chemotherapeutic agent. In someembodiments, the one or more additional agents for treatment or therapyis an immunomodulatory agent, immune checkpoint inhibitor, adenosinepathway or adenosine receptor antagonist or agonist and kinaseinhibitors. In some embodiments, the combination treatment orcombination therapy includes an additional treatment, such as a surgicaltreatment, transplant, and/or radiation therapy.

In some embodiments, the additional agent is selected from among aprotein phosphatase inhibitor, a kinase inhibitor, a cytokine, animmunomodulator, or an agent that decreases the level or activity of aregulatory T (Treg) cell. In some embodiments, the additional agentenhances safety, by virtue of reducing or ameliorating adverse effectsof the administered cell therapy. In some embodiments, the additionalagent can treat the same disease, condition or a comorbidity. In someembodiments, the additional agent can ameliorate, reduce or eliminateone or more toxicities, adverse effects or side effects that areassociated with administration of the cells, e.g., CAR-expressing cells.

In some embodiments, the additional therapy, treatment or agent includeschemotherapy, radiation therapy, surgery, transplantation, adoptive celltherapy, antibodies, cytotoxic agents, chemotherapeutic agents,cytokines, growth inhibitory agents, anti-hormonal agents, kinaseinhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatoryagents, immunosuppressive agents, immune checkpoint inhibitors,antibiotics, angiogenesis inhibitors, metabolic modulators or othertherapeutic agents or any combination thereof. In some embodiments, theadditional agent is a protein, a peptide, a nucleic acid, a smallmolecule agent, a cell, a toxin, a lipid, a carbohydrate or combinationsthereof, or any other type of therapeutic agent, e.g. radiation. In someembodiments, the additional therapy, agent or treatment includessurgery, chemotherapy, radiation therapy, transplantation,administration of cells expressing a recombinant receptor, e.g., CAR,kinase inhibitor, immune checkpoint inhibitor, mTOR pathway inhibitor,immunosuppressive agents, immunomodulators, antibodies, immunoablativeagents, antibodies and/or antigen binding fragments thereof, antibodyconjugates, other antibody therapies, cytotoxins, steroids, cytokines,peptide vaccines, hormone therapy, antimetabolites, metabolicmodulators, drugs that inhibit either the calcium dependent phosphatasecalcineurin or the p70S6 kinase FK506) or inhibit the p70S6 kinase,alkylating agents, anthracyclines, vinca alkaloids, proteosomeinhibitors, GITR agonists, protein tyrosine phosphatase inhibitors,protein kinase inhibitors, an oncolytic virus, and/or other types ofimmunotherapy. In some embodiments, the additional agent or treatment isbone marrow transplantation, T cell ablative therapy using chemotherapyagents such as, fludarabine, external-beam radiation therapy (XRT),cyclophosphamide, and/or antibody therapy.

In some embodiments, the additional agent is a kinase inhibitor, e.g.,an inhibitor of Bruton's tyrosine kinase (Btk), e.g., ibrutinib. In someembodiments, the additional agent is an adenosine pathway or adenosinereceptor antagonist or agonist. In some embodiments, the additionalagent is an immunomodulator such as thalidomide or a thalidomidederivative (e.g., lenalidomide). In some embodiments, the additionaltherapy, agent or treatment is a cytotoxic or chemotherapy agent, abiologic therapy (e.g., antibody, e.g., monoclonal antibody, or cellulartherapy), or an inhibitor (e.g., kinase inhibitor).

In some embodiments, the additional agent is a chemotherapeutic agent.Exemplary chemotherapeutic agents include an anthracycline (e.g.,doxorubicin, such as liposomal doxorubicin); a vinca alkaloid (e.g.,vinblastine, vincristine, vindesine, vinorelbine); an alkylating agent(e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide,temozolomide); an immune cell antibody (e.g., alemtuzamab, gemtuzumab,rituximab, tositumomab); an antimetabolite (including, e.g., folic acidantagonists, pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors such as fludarabine); a TNFR glucocorticoid induced TNFRrelated protein (GITR) agonist; a proteasome inhibitor (e.g.,aclacinomycin A, gliotoxin or bortezomib); an immunomodulatory such asthalidomide or a thalidomide derivative (e.g., lenalidomide).

In some embodiments, the additional agent is an immunomodulatory agent.In some embodiments, the combination therapy includes animmunomodulatory agent that can stimulate, amplify and/or otherwiseenhance an anti-tumor immune response, e.g. anti-tumor immune responsefrom the administered engineered cells, such as by inhibitingimmunosuppressive signaling or enhancing immunostimulant signaling. Insome embodiments, the immunomodulatory agent is a peptide, protein or isa small molecule. In some embodiments, the protein can be a fusionprotein or a recombinant protein. In some embodiments, theimmunomodulatory agent binds to an immunologic target, such as a cellsurface receptor expressed on immune cells, such a T cells, B cells orantigen-presenting cells. For example, in some embodiments, theimmunomodulatory agent is an antibody or antigen-binding antibodyfragment, a fusion protein, a small molecule or a polypeptide. In someembodiments, the binding molecules, recombinant receptors, cells and/orcompositions are administered in combination with an additional agentthat is an antibody or an antigen-binding fragment thereof, such as amonoclonal antibody.

In some embodiments, the immunomodulatory agent blocks, inhibits orcounteracts a component of the immune checkpoint pathway. The immunesystem has multiple inhibitory pathways that are involved in maintainingself-tolerance and for modulating immune responses. Tumors can usecertain immune-checkpoint pathways as a major mechanism of immuneresistance, particularly against T cells that are specific for tumorantigens (Pardoll (2012) Nature Reviews Cancer 12:252-264), e.g.,engineered cells such as CAR-expressing cells. Because many such immunecheckpoints are initiated by ligand-receptor interactions, they can bereadily blocked by antibodies against the ligands and/or theirreceptors. In contrast to the majority of anti-cancer agents, checkpointinhibitors do not necessarily target tumor cells directly, but rathertarget lymphocyte receptors or their ligands in order to enhance theendogenous antitumor activity of the immune system.

In some embodiments, the additional agent is an immunomodulatory agentthat is an antagonist molecule or is an immune checkpoint inhibitorcapable of inhibiting or blocking a function of a molecule, or signalingpathway, involving an immune checkpoint molecule. In some embodiments,the immune checkpoint molecule or pathway is PD-1, PD-L1, PD-L2, CTLA-4,LAG-3, TIM3, VISTA, adenosine 2A Receptor (A2AR), or adenosine or apathway involving any of the foregoing. In certain embodiments,antagonistic molecules blocking an immune checkpoint pathway, such assmall molecules, nucleic acid inhibitors (e.g., RNAi) or antibodymolecules, are becoming promising avenues of immunotherapy for cancerand other diseases.

In some embodiments, the immune checkpoint inhibitor is a molecule thattotally or partially reduces, inhibits, interferes with or modulate oneor more checkpoint proteins. Checkpoint proteins regulate T-cellactivation or function. These proteins are responsible forco-stimulatory or inhibitory interactions of T-cell responses. Immunecheckpoint proteins regulate and maintain self-tolerance and theduration and amplitude of physiological immune responses.

Immune checkpoint inhibitors include any agent that blocks or inhibitsin a statistically significant manner, the inhibitory pathways of theimmune system. Such inhibitors may include small molecule inhibitors ormay include antibodies, or antigen binding fragments thereof, that bindto and block or inhibit immune checkpoint receptors, ligands and/orreceptor-ligand interaction. In some embodiments, modulation,enhancement and/or stimulation of particular receptors can overcomeimmune checkpoint pathway components. Illustrative immune checkpointmolecules that may be targeted for blocking, inhibition, modulation,enhancement and/or stimulation include, but are not limited to, PD-1(CD279), PD-L1 (CD274, B7-H1), PDL2 (CD273, B7-DC), CTLA-4, LAG-3(CD223), TIM-3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR),CD40, OX40 (CD134, TNFRSF4), CXCR2, tumor associated antigens (TAA),B7-H3, B7-H4, BTLA, HVEM, GAL9, B7H3, B7H4, VISTA, KIR, 2B4 (belongs tothe CD2 family of molecules and is expressed on all NK, γδ, and memoryCD8⁺ (αβ) T cells), CD160 (also referred to as BY55), CGEN-15049, CEACAM(e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), TIGIT, LAIR1, CD160, 2B4,CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR,A2aR, MEW class I, MHC class II, GAL9, adenosine, and a transforminggrowth factor receptor (TGFR; e.g., TGFR beta). Immune checkpointinhibitors include antibodies, or antigen binding fragments thereof, orother binding proteins, that bind to and block or inhibit and/or enhanceor stimulate the activity of one or more of any of the said molecules.

Exemplary immune checkpoint inhibitors include Tremelimumab (CTLA-4blocking antibody, also known as ticilimumab, CP-675,206), anti-OX40,PD-L1 monoclonal antibody (Anti-B7-H1; MEDI4736), MK-3475 (PD-1blocker), nivolumab (anti-PD-1 antibody), CT-011 (anti-PD-1 antibody),BY55 monoclonal antibody, AMP224 (anti-PD-L1 antibody), BMS-936559(anti-PD-L1 antibody), MPLDL3280A (anti-PD-L1 antibody), MSB0010718C(anti-PD-L1 antibody) and ipilimumab (anti-CTLA-4 antibody, also knownas Yervoy®, MDX-010 and MDX-101). Exemplary of immunomodulatoryantibodies include, but are not limited to, Daclizumab (Zenapax),Bevacizumab (Avastin®), Basiliximab, Ipilimumab, Nivolumab,pembrolizumab, MPDL3280A, Pidilizumab (CT-011), MK-3475, BMS-936559,MPDL3280A (Atezolizumab), tremelimumab, IMP321, BMS-986016, LAG525,urelumab, PF-05082566, TRX518, MK-4166, dacetuzumab (SGN-40),lucatumumab (HCD122), SEA-CD40, CP-870, CP-893, MEDI6469, MEDI6383,MOXR0916, AMP-224, MSB0010718C (Avelumab), MEDI4736, PDR001, rHIgM12B7,Ulocuplumab, BKT140, Varlilumab (CDX-1127), ARGX-110, MGA271, lirilumab(BMS-986015, IPH2101), IPH2201, ARGX-115, Emactuzumab, CC-90002 andMNRP1685A or an antibody-binding fragment thereof. Other exemplaryimmunomodulators include, e.g., afutuzumab (available from Roche®);pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®);thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of humancytokines including interleukin 1, interleukin 2, and interferon.gamma.,CAS 951209-71-5, available from IRX Therapeutics).

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that binds to and/or inhibitsProgrammed cell death 1 (PD-1). PD-1 is an immune checkpoint proteinthat is expressed in B cells, NK cells, and T cells (Shinohara et al.,1995, Genomics 23:704-6; Blank et al., 2007, Cancer Immunol Immunother56:739-45; Finger et al., 1997, Gene 197:177-87; Pardoll (2012) NatureReviews Cancer 12:252-264). The major role of PD-1 is to limit theactivity of T cells in peripheral tissues during inflammation inresponse to infection, as well as to limit autoimmunity. PD-1 expressionis induced in activated T cells and binding of PD-1 to one of itsendogenous ligands acts to inhibit T-cell activation by inhibitingstimulatory kinases. PD-1 also acts to inhibit the TCR “stop signal”.PD-1 is highly expressed on Treg cells and may increase theirproliferation in the presence of ligand (Pardoll (2012) Nature ReviewsCancer 12:252-264). Anti-PD 1 antibodies have been used for treatment ofmelanoma, non-small-cell lung cancer, bladder cancer, prostate cancer,colorectal cancer, head and neck cancer, triple-negative breast cancer,leukemia, lymphoma and renal cell cancer (Topalian et al., 2012, N EnglJ Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Bergeret al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013,Oral Oncol 49:1089-96; Menzies & Long, 2013, Ther Adv Med Oncol5:278-85). Exemplary anti-PD-1 antibodies include nivolumab (Opdivo byBMS), pembrolizumab (Keytruda by Merck), pidilizumab (CT-011 by CureTech), lambrolizumab (MK-3475 by Merck), and AMP-224 (Merck), nivolumab(also referred to as Opdivo, BMS-936558 or MDX1106; Bristol-MyersSquibb) is a fully human IgG4 monoclonal antibody which specificallyblocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodiesthat specifically bind to PD-1 are described in U.S. Pat. No. 8,008,449and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1kmonoclonal antibody that binds to PD-1. Pidilizumab and other humanizedanti-PD-1 monoclonal antibodies are described in WO2009/101611.Pembrolizumab (formerly known as lambrolizumab, and also referred to asKeytruda, MK03475; Merck) is a humanized IgG4 monoclonal antibody thatbinds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodiesare described in U.S. Pat. No. 8,354,509 and WO2009/114335. Otheranti-PD-1 antibodies include AMP 514 (Amplimmune), among others, e.g.,anti-PD-1 antibodies described in U.S. Pat. No. 8,609,089, US2010028330, US 20120114649 and/or US 20150210769. AMP-224 (B7-DCIg;Amplimmune; e.g., described in WO2010/027827 and WO2011/066342), is aPD-L2 Fc fusion soluble receptor that blocks the interaction betweenPD-1 and B7-H1.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that binds to or inhibits PD-L1(also known as CD274 and B7-H1) and/or PD-L2 (also known as CD273 andB7-DC). PD-L1 and PD-L2 are ligands for PD-1, found on activated Tcells, B cells, myeloid cells, macrophages, and some types of tumorcells. Anti-tumor therapies have focused on anti-PD-L1 antibodies. Thecomplex of PD-1 and PD-L1 inhibits proliferation of CD8⁺ T cells andreduces the immune response (Topalian et al., 2012, N Engl J Med366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65). Anti-PD-L1antibodies have been used for treatment of non-small cell lung cancer,melanoma, colorectal cancer, renal-cell cancer, pancreatic cancer,gastric cancer, ovarian cancer, breast cancer, and hematologicmalignancies (Brahmer et al., 2012, N Eng J Med 366:2455-65; Ott et al.,2013, Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger etal., 2008, Clin Cancer Res 14:13044-51). Exemplary anti-PD-L1 antibodiesinclude MDX-1105 (Medarex), MEDI4736 (Medimmune) MPDL3280A (Genentech),BMS-935559 (Bristol-Myers Squibb) and MSB0010718C MEDI4736 (Medimmune)is a human monoclonal antibody that binds to PD-L1, and inhibitsinteraction of the ligand with PD-1. MDPL3280A (Genentech/Roche) is ahuman Fc optimized IgG1 monoclonal antibody that binds to PD-L1.MDPL3280A and other human monoclonal antibodies to PD-L1 are describedin U.S. Pat. No. 7,943,743 and U.S Publication No. 20120039906. Otheranti-PD-L1 binding agents include YW243.55.570 (see WO2010/077634) andMDX-1105 (also referred to as BMS-936559, and, e.g., anti-PD-L1 bindingagents described in WO2007/005874).

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that is an inhibitor ofCytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152,or binds to CTLA-4. CTLA-4 is a co-inhibitory molecule that functions toregulate T-cell activation. CTLA-4 is a member of the immunoglobulinsuperfamily that is expressed exclusively on T-cells. CTLA-4 acts toinhibit T-cell activation and is reported to inhibit helper T-cellactivity and enhance regulatory T-cell immunosuppressive activity.Although the precise mechanism of action of CTLA-4 remains underinvestigation, it has been suggested that it inhibits T cell activationby outcompeting CD28 in binding to CD80 and CD86, as well as activelydelivering inhibitor signals to the T cell (Pardoll (2012) NatureReviews Cancer 12:252-264). Anti-CTLA-4 antibodies have been used inclinical trials for the treatment of melanoma, prostate cancer, smallcell lung cancer, non-small cell lung cancer (Robert & Ghiringhelli,2009, Oncologist 14:848-61; Ott et al., 2013, Clin Cancer Res 19:5300;Weber, 2007, Oncologist 12:864-72; Wada et al., 2013, J Transl Med11:89). A significant feature of anti-CTLA-4 is the kinetics ofanti-tumor effect, with a lag period of up to 6 months after initialtreatment required for physiologic response. In some cases, tumors mayactually increase in size after treatment initiation, before a reductionis seen (Pardoll (2012) Nature Reviews Cancer 12:252-264). Exemplaryanti-CTLA-4 antibodies include ipilimumab (Bristol-Myers Squibb) andtremelimumab (Pfizer). Ipilimumab has recently received FDA approval fortreatment of metastatic melanoma (Wada et al., 2013, J Transl Med11:89).

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that bind to and/or inhibitsLymphocyte activation gene-3 (LAG-3), also known as CD223. LAG-3 isanother immune checkpoint protein. LAG-3 has been associated with theinhibition of lymphocyte activity and in some cases the induction oflymphocyte anergy. LAG-3 is expressed on various cells in the immunesystem including B cells, NK cells, and dendritic cells. LAG-3 is anatural ligand for the MHC class II receptor, which is substantiallyexpressed on melanoma-infiltrating T cells including those endowed withpotent immune-suppressive activity. Exemplary anti-LAG-3 antibodiesinclude BMS-986016 (Bristol-Myers Squib), which is a monoclonal antibodythat targets LAG-3. IMP701 (Immutep) is an antagonist LAG-3 antibody andIMP731 (Immutep and GlaxoSmithKline) is a depleting LAG-3 antibody.Other LAG-3 inhibitors include IMP321 (Immutep), which is a recombinantfusion protein of a soluble portion of LAG-3 and Ig that binds to MHCclass II molecules and activates antigen presenting cells (APC). Otherantibodies are described, e.g., in WO2010/019570 and US 2015/0259420.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that bins to and/or inhibitsT-cell immunoglobulin domain and mucin domain-3 (TIM-3). TIM-3 wasinitially identified on activated Th1 cells, has been shown to be anegative regulator of the immune response. Blockade of TIM-3 promotesT-cell mediated anti-tumor immunity and has anti-tumor activity in arange of mouse tumor models. Combinations of TIM-3 blockade with otherimmunotherapeutic agents such as TSR-042, anti-CD137 antibodies andothers, can be additive or synergistic in increasing anti-tumor effects.TIM-3 expression has been associated with a number of different tumortypes including melanoma, NSCLC and renal cancer, and additionally,expression of intratumoral TIM-3 has been shown to correlate with poorprognosis across a range of tumor types including NSCLC, cervical, andgastric cancers. Blockade of TIM-3 is also of interest in promotingincreased immunity to a number of chronic viral diseases. TIM-3 has alsobeen shown to interact with a number of ligands including galectin-9,phosphatidylserine and HMGB1, although which of these, if any, arerelevant in regulation of anti-tumor responses is not clear at present.In some embodiments, antibodies, antibody fragments, small molecules, orpeptide inhibitors that target TIM-3 can bind to the IgV domain of TIM-3to inhibit interaction with its ligands. Exemplary antibodies andpeptides that inhibit TIM-3 are described in US 2015/0218274,WO2013/006490 and US 2010/0247521. Other anti-TIM-3 antibodies includehumanized versions of RMT3-23 (Ngiow et al., 2011, Cancer Res,71:3540-3551), and clone 8B.2C12 (Monney et al., 2002, Nature,415:536-541). Bi-specific antibodies that inhibit TIM-3 and PD-1 aredescribed in US 2013/0156774.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that is a CEACAM inhibitor(e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor). In someembodiments, the inhibitor of CEACAM is an anti-CEACAM antibodymolecule. Exemplary anti-CEACAM-1 antibodies are described in WO2010/125571, WO 2013/082366 WO 2014/059251 and WO 2014/022332, e.g., amonoclonal antibody 34B1, 26H7, and 5F4; or a recombinant form thereof,as described in, e.g., US 2004/0047858, U.S. Pat. No. 7,132,255 and WO99/052552. In some embodiments, the anti-CEACAM antibody binds toCEACAM-5 as described in, e.g., Zheng et al. PLoS One. (2011) 6(6):e21146), or crossreacts with CEACAM-1 and CEACAM-5 as described in,e.g., WO 2013/054331 and US 2014/0271618.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that binds to and/or inhibits4-1BB, also known as CD137. 4-1BB is a transmembrane glycoproteinbelonging to the TNFR superfamily. 4-1BB receptors are present onactivated T cells and B cells and monocytes. An exemplary anti-4-1BBantibody is urelumab (BMS-663513), which has potential immunostimulatoryand antineoplastic activities.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that binds to and/or inhibitsTumor necrosis factor receptor superfamily, member 4 (TNFRSF4), alsoknown as OX40 and CD134. TNFRSF4 is another member of the TNFRsuperfamily. OX40 is not constitutively expressed on resting naïve Tcells and acts as a secondary co-stimulatory immune checkpoint molecule.Exemplary anti-OX40 antibodies are MEDI6469 and MOXR0916 (RG7888,Genentech).

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent or a molecule that decreasesthe regulatory T cell (Treg) population. Methods that decrease thenumber of (e.g., deplete) Treg cells are known in the art and include,e.g., CD25 depletion, cyclophosphamide administration, and modulatingGlucocorticoid-induced TNFR family related gene (GITR) function. GITR isa member of the TNFR superfamily that is upregulated on activated Tcells, which enhances the immune system. Reducing the number of Tregcells in a subject prior to apheresis or prior to administration ofengineered cells, e.g., CAR-expressing cells, can reduce the number ofunwanted immune cells (e.g., Tregs) in the tumor microenvironment andreduces the subject's risk of relapse. In some embodiments, theadditional agent includes a molecule targeting GITR and/or modulatingGITR functions, such as a GITR agonist and/or a GITR antibody thatdepletes regulatory T cells (Tregs). In some embodiments, the additionalagent includes cyclophosphamide. In some embodiments, the GITR bindingmolecule and/or molecule modulating GITR function (e.g., GITR agonistand/or Treg depleting GITR antibodies) is administered prior to theengineered cells, e.g., CAR-expressing cells. For example, in someembodiments, the GITR agonist can be administered prior to apheresis ofthe cells. In some embodiments, cyclophosphamide is administered to thesubject prior to administration (e.g., infusion or re-infusion) of theengineered cells, e.g., CAR-expressing cells or prior to apheresis ofthe cells. In some embodiments, cyclophosphamide and an anti-GITRantibody are administered to the subject prior to administration (e.g.,infusion or re-infusion) of the engineered cells, e.g., CAR-expressingcells or prior to apheresis of the cells.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that is a GITR agonist.Exemplary GITR agonists include, e.g., GITR fusion proteins andanti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as,e.g., a GITR fusion protein described in U.S. Pat. No. 6,111,090,European Patent No. 090505B 1, U.S. Pat. No. 8,586,023, PCT PublicationNos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibodydescribed, e.g., in U.S. Pat. No. 7,025,962, European Patent No.1947183B 1, U.S. Pat. Nos. 7,812,135, 8,388,967, 8,591,886, EuropeanPatent No. EP 1866339, PCT Publication No. WO 2011/028683, PCTPublication No. WO 2013/039954, PCT Publication No. WO2005/007190, PCTPublication No. WO 2007/133822, PCT Publication No. WO2005/055808, PCTPublication No. WO 99/40196, PCT Publication No. WO 2001/03720, PCTPublication No. WO99/20758, PCT Publication No. WO2006/083289, PCTPublication No. WO 2005/115451, U.S. Pat. No. 7,618,632, and PCTPublication No. WO 2011/051726. An exemplary anti-GITR antibody isTRX518.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, enhances tumor infiltration ortransmigration of the administered cells, e.g., CAR-expressing cells.For example, in some embodiments, the additional agent stimulates CD40,such as CD40L, e.g., recombinant human CD40L. Cluster of differentiation40 (CD40) is also a member of the TNFR superfamily. CD40 is acostimulatory protein found on antigen-presenting cells and mediates abroad variety of immune and inflammatory responses. CD40 is alsoexpressed on some malignancies, where it promotes proliferation.Exemplary anti-CD40 antibodies are dacetuzumab (SGN-40), lucatumumab(Novartis, antagonist), SEA-CD40 (Seattle Genetics), and CP-870,893. Insome embodiments, the additional agent that enhances tumor infiltrationincludes tyrosine kinase inhibitor sunitnib, heparanase, and/orchemokine receptors such as CCR2, CCR4, and CCR7.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an immunomodulatory agent that is astructural or functional analog or derivative of thalidomide and/or aninhibitor of E3 ubiquitin ligase. In some embodiments, theimmunomodulatory agent binds to cereblon (CRBN). In some embodiments,the immunomodulatory agent binds to the CRBN E3 ubiquitin-ligasecomplex. In some embodiments, the immunomodulatory agent binds to CRBNand the CRBN E3 ubiquitin-ligase complex. In some embodiments, theimmunomodulatory agent up-regulates the protein or gene expression ofCRBN. In some aspects, CRBN is the substrate adaptor for the CRL4^(CRBN)E3 ubiquitin ligase, and modulates the specificity of the enzyme. Insome embodiments, binding to CRB or the CRBN E3 ubiquitin ligase complexinhibits E3 ubiquitin ligase activity. In some embodiments, theimmunomodulatory agent induces the ubiqutination of KZF1 (Ikaros) andIKZF3 (Aiolos) and/or induces degradation of IKZF1 (Ikaros) and IKZF3(Aiolos). In some embodiments, the immunomodulatory agent induces theubiquitination of casein kinase 1A1 (CK1α) by the CRL4^(CRBN) E3ubiquitin ligase. In some embodiments, the ubiquitination of CK1αresults in CK1α degradation.

In some embodiments, the immunomodulatory agent is an inhibitor of theIkaros (IKZF1) transcription factor. In some embodiments, theimmunomodulatory agent enhances ubiquitination of Ikaros. In someembodiments, the immunomodulatory agent enhances the degradation ofIkaros. In some embodiments, the immunomodulatory agent down-regulatesthe protein or gene expression of Ikaros. In some embodiments,administration of the immunomodulatory agent causes a decrease in Ikarosprotein levels.

In some embodiments, the immunomodulatory agent is an inhibitor of theAiolos (IKZF3) transcription factor. In some embodiments, theimmunomodulatory agent enhances ubiquitination of Aiolos. In someembodiments, the immunomodulatory agent enhances the degradation ofAiolos. In some embodiments, the immunomodulatory agent down-regulatesthe protein or gene expression of Aiolos. In some embodiments,administration of the immunomodulatory agent causes a decrease in Aiolosprotein levels.

In some embodiments, the immunomodulatory agent is an inhibitor of boththe Ikaros (IKZF1) and Aiolos (IKZF3) transcription factors. In someembodiments, the immunomodulatory agent enhances ubiquitination of bothIkaros and Aiolos. In some embodiments, the immunomodulatory agentenhances the degradation of both Ikaros and Aiolos. In some embodiments,the immunomodulatory agent enhances ubiquitination and degradation ofboth Ikaros and Aiolos. In some embodiments, administration of theimmunomodulatory agent causes both Aiolos protein levels and Ikarosprotein levels to decrease.

In some embodiments, the immunomodulatory agent is a selective cytokineinhibitory drug (SelCID). In some embodiments, the immunomodulatoryagent inhibits the activity of phosphodiesterase-4 (PDE4). In someembodiments, the immunomodulatory agent suppresses the enzymaticactivity of the CDC25 phosphatases. In some embodiments, theimmunomodulatory agent alters the intracellular trafficking of CDC25phosphatases.

In some embodiments, the immunomodulatory agent is thalidomide(242,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione) or an analog orderivative of thalidomide. In certain embodiments, a thalidomidederivative includes structural variants of thalidomide that have asimilar biological activity. Exemplary thalidomide derivatives include,but are not limited to lenalidomide (REVLIMMUNOMODULATORY COMPOUND™;Celgene Corporation), pomalidomide (also known as ACTIMMUNOMODULATORYCOMPOUND™ or POMALYST™ (Celgene Corporation)), CC-1088, CDC-501, andCDC-801, and the compounds disclosed in U.S. Pat. Nos. 5,712,291;7,320,991; and 8,716,315; U.S. Appl. No. 2016/0313300; and PCT Pub. Nos.WO 2002/068414 and WO 2008/154252.

In some embodiments, the immunomodulatory agent is 1-oxo- and 1,3dioxo-2-(2,6-dioxopiperldin-3-yl) isoindolines substituted with amino inthe benzo ring as described in U.S. Pat. No. 5,635,517 which isincorporated herein by reference.

In some embodiments, the immunomodulatory agent is a compound of thefollowing formula:

wherein one of X and Y is —C(O)— and the other of X and Y is —C(O)— or—CH₂—, and R⁵ is hydrogen or lower alkyl, or a pharmaceuticallyacceptable salt thereof. In some embodiments, X is —C(O)— and Y is—CH₂—. In some embodiments, both X and Y are —C(O)—. In someembodiments, R⁵ is hydrogen. In other embodiments, R⁵ is methyl.

In some embodiments, the immunomodulatory compound is a compound thatbelongs to a class of substituted 2-(2,6-dioxopiperidin-3-yl)phthalimmunomodulatory compounds and substituted2-(2,6-dioxopiperldin-3-yl)-1-oxoisoindoles, such as those described inU.S. Pat. Nos. 6,281,230; 6,316,471; 6,335,349; and 6,476,052, andInternational Patent Application No. PCT/US97/13375 (InternationalPublication No. WO 98/03502), each of which is incorporated herein byreference.

In some embodiments, the immunomodulatory agent is a compound of thefollowing formula:

wherein

one of X and Y is —C(O)— and the other of X and Y is —C(O)— or —CH₂—;

(1) each of R¹, R², R³, and R⁴ are independently halo, alkyl of 1 to 4carbon atoms, or alkoxy or 1 to 4 carbon atoms, or

(2) one of R¹, R³, R⁴, and R⁵ is —NHR^(a) and the remaining of R¹, R²,R³, and R⁴ is are hydrogen, wherein R^(a) is hydrogen or alkyl of 1 to 8carbon atoms;

R⁵ is hydrogen or alkyl of 1 to 8 carbon atoms, benzyl, or halo;

provided that R⁵ is other than hydrogen if X and Y are —C(O)— and (i)each of R¹, R², R³, and R⁴ is fluoro; or (ii) one of R¹, R², R³, and R⁴is amino;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the immunomodulatory agent is a compound thatbelongs to a class of isoindole-immunomodulatory compounds disclosed inU.S. Pat. No. 7,091,353, U.S. Patent Publication No. 2003/0045552, andInternational Application No. PCT/USOI/50401 (International PublicationNo. WO02/059106), each of which are incorporated herein by reference.For example, in some embodiments, the immunomodulatory agent is[2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl]-amide;(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-carbamicacid tert-butyl ester;4-(aminomethyl)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione;N-(2-(2,6-dioxo-piperidin-3-yl)-1,3-dioxo-2,3-dihydro-1H-isoindol-4-ylmethyl)-acetamide;N-{(2-(2,6-dioxo(3-piperidyl)-1,3-dioxoisoindolin-4-yl)methyl}cyclopropyl-carboxamide;2-chloro-N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}acetamide;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-3-pyridylcarboxamide;3-{1-oxo-4-(benzylamino)isoindolin-2-yl}piperidine-2,6-dione;2-(2,6-dioxo(3-piperidyl))-4-(benzylamino)isoindoline-1,3-dione;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}propanamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-3-pyridylcarboxamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}heptanamide;N-{(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)methyl}-2-furylcarboxamide;{N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)carbamoyl}methylacetate;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)pentanamide;N-(2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl)-2-thienylcarboxamide;N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(butylamino)carboxamide;N-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(octylamino)carboxamide;orN-{[2-(2,6-dioxo(3-piperidyl))-1,3-dioxoisoindolin-4-yl]methyl}(benzylamino)carboxamide.

In some embodiments, the immunomodulatory agent is a compound thatbelongs to a class of isoindole-immunomodulatory compounds disclosed inU.S. Patent Application Publication Nos. 2002/0045643, InternationalPublication No. WO 98/54170, and U.S. Pat. No. 6,395,754, each of whichis incorporated herein by reference. In some embodiments, theimmunomodulatory agent is a tetra substituted2-(2,6-dioxopiperdin-3-yl)-1-oxoisoindolines described in U.S. Pat. No.5,798,368, which is incorporated herein by reference. In someembodiments, the immunomodulatory agent is 1-oxo and1,3-dioxo-2-(2,6-dioxopiperidin-3-yl) isoindolines disclosed in U.S.Pat. No. 6,403,613, which is incorporated herein by reference. In someembodiments the immunomodulatory agent is a 1-oxo or1,3-dioxoisoindoline substituted in the 4- or 5-position of the indolinering as described in U.S. Pat. Nos. 6,380,239 and 7,244,759, both ofwhich are incorporated herein by reference.

In some embodiments, the immunomodulatory agent is2-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric acid or4-(4-amino-1-oxo-1,3-dihydro-isoindol-2-yl)-4-carbamoyl-butyric acid. Insome embodiments, the immunomodulatory compound is4-carbamoyl-4-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-butyricacid,4-carbamoyl-2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-butyricacid,2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-4-phenylcarbamoyl-butyricacid, or2-{4-[(furan-2-yl-methyl)-amino]-1,3-dioxo-1,3-dihydro-isoindol-2-yl}-pentanedioicacid.

In some embodiments, the immunomodulatory agent is a isoindoline-1-oneor isoindoline-1,3-dione substituted in the 2-position with2,6-dioxo-3-hydroxypiperidin-5-yl as described in U.S. Pat. No.6,458,810, which is incorporated herein by reference. In someembodiments, the immunomodulatory compound is3-(5-amino-2-methyl-4-oxo-4H-quinazolin-3-yl)-piperidine-2,6-dione, oran enantiomer or a mixture of enantiomers thereof; or a pharmaceuticallyacceptable salt, solvate, hydrate, co-crystal, clathrate, or polymorphthereof. In some embodiments, the immunomodulatory compound is3-[4-(4-morpholin-4-ylmethyl-benzyloxy)-1-oxo-1,3-dihydro-isoindol-2-yl]-piperidine-2,6-dione.

In some embodiments, the immunomodulatory agent is as described inOshima, K. et al., Nihon Rinsho., 72(6):1130-5 (2014); Millrine, D. etal., Trends Mol Med., 23(4):348-364 (2017); and Collins, et al., BiochemJ., 474(7):1127-1147 (2017).

In some embodiments, the immunomodulatory agent is lenalidomide,pomalidomide, avadomide, a stereoisomer of lenalidomide, pomalidomide,avadomide or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof. In some embodiments, theimmunomodulatory compound is lenalidomide, a stereoisomer oflenalidomide or a pharmaceutically acceptable salt, solvate, hydrate,co-crystal, clathrate, or polymorph thereof. In some embodiments, theimmunomodulatory compound is lenalidomide, or((RS)-3-(4-Amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione).

In some embodiments, the additional agent includes thalidomide drugs oranalogs thereof and/or derivatives thereof, such as lenalidomide,pomalidomide or apremilast. See, e.g., Bertilaccio et al., Blood (2013)122:4171, Otahal et al., Oncoimmunology (2016) 5(4):e1115940; Fecteau etal., Blood (2014) 124(10):1637-1644 and Kuramitsu et al., Cancer GeneTherapy (2015) 22:487-495). Lenalidomide((RS)-3-(4-Amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione;also known as Revlimid) is a synthetic derivative of thalidomide, andhas multiple immunomodulatory effects, including enforcement of immunesynapse formation between T cell and antigen presenting cells (APCs).For example, in some cases, lenalidomide modulates T cell responses andresults in increased interleukin (IL)-2 production in CD4⁺ and CD8⁺ Tcells, induces the shift of T helper (Th) responses from Th2 to Th1,inhibits expansion of regulatory subset of T cells (Tregs), and improvesfunctioning of immunological synapses in follicular lymphoma and chroniclymphocytic leukemia (CLL) (Otahal et al., Oncoimmunology (2016)5(4):e1115940). Lenalidomide also has direct tumoricidal activity inpatients with multiple myeloma (MM) and directly and indirectlymodulates survival of CLL tumor cells by affecting supportive cells,such as nurse-like cells found in the microenvironment of lymphoidtissues. Lenalidomide also can enhance T-cell proliferation andinterferon-γ production in response to activation of T cells via CD3ligation or dendritic cell-mediated activation. Lenalidomide can alsoinduce malignant B cells to express higher levels of immunostimulatorymolecules such as CD80, CD86, HLA-DR, CD95, and CD40 (Fecteau et al.,Blood (2014) 124(10):1637-1644). In some embodiments, lenalidomide isadministered at a dosage of from about 1 mg to about 20 mg daily, e.g.,from about 1 mg to about 10 mg, from about 2.5 mg to about 7.5 mg, fromabout 5 mg to about 15 mg, such as about 5 mg, 10 mg, 15 mg or 20 mgdaily. In some embodiments, lenalidomide is administered at a dose offrom about 10 μg/kg to 5 mg/kg, e.g., about 100 μg/kg to about 2 mg/kg,about 200 μg/kg to about 1 mg/kg, about 400 μg/kg to about 600 μg/kg,such as about 500 μg/kg.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is a B-cell inhibitor. In some embodiments,the additional agent is one or more B-cell inhibitors selected fromamong inhibitors of CD10, CD19, CD20, CD22, CD34, CD123, CD79a, CD79b,CD179b, FLT-3, or ROR1, or a combination thereof. In some embodiments,the B-cell inhibitor is an antibody (e.g., a mono- or bispecificantibody) or an antigen binding fragment thereof. In some embodiments,the additional agent is an engineered cell expressing recombinantreceptors that target B-cell targets, e.g., CD10, CD19, CD20, CD22,CD34, CD123, CD79a, CD79b, CD179b, FLT-3, or ROR1.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is a CD20 inhibitor, e.g., an anti-CD20antibody (e.g., an anti-CD20 mono- or bi-specific antibody) or afragment thereof. Exemplary anti-CD20 antibodies include but are notlimited to rituximab, ofatumumab, ocrelizumab (also known as GA101 orRO5072759), veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals),ocaratuzumab (also known as AME-133v or ocaratuzumab), and Pro131921(Genentech). See, e.g., Lim et al. Haematologica. (2010) 95(1):135-43.In some embodiments, the anti-CD20 antibody comprises rituximab.Rituximab is a chimeric mouse/human monoclonal antibody IgG1 kappa thatbinds to CD20 and causes cytolysis of a CD20 expressing cell. In someembodiments, the additional agent includes rituximab. In someembodiments, the CD20 inhibitor is a small molecule.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is a CD22 inhibitor, e.g., an anti-CD22antibody (e.g., an anti-CD22 mono- or bi-specific antibody) or afragment thereof. Exemplary anti-CD22 antibodies include epratuzumab andRFB4. In some embodiments, the CD22 inhibitor is a small molecule. Insome embodiments, the antibody is a monospecific antibody, optionallyconjugated to a second agent such as a chemotherapeutic agent. Forinstance, in some embodiments, the antibody is an anti-CD22 monoclonalantibody-MMAE conjugate (e.g., DCDT2980S). In some embodiments, theantibody is an scFv of an anti-CD22 antibody, e.g., an scFv of antibodyRFB4. In some embodiments, the scFv is fused to all of or a fragment ofPseudomonas exotoxin-A (e.g., BL22). In some embodiments, the scFv isfused to all of or a fragment of (e.g., a 38 kDa fragment of)Pseudomonas exotoxin-A (e.g., moxetumomab pasudotox). In someembodiments, the anti-CD22 antibody is an anti-CD19/CD22 bispecificantibody, optionally conjugated to a toxin. For instance, in someembodiments, the anti-CD22 antibody comprises an anti-CD19/CD22bispecific portion, (e.g., two scFv ligands, recognizing human CD19 andCD22) optionally linked to all of or a portion of diphtheria toxin (DT),e.g., first 389 amino acids of diphtheria toxin (DT), DT 390, e.g., aligand-directed toxin such as DT2219ARL). In some embodiments, thebispecific portion (e.g., anti-CD 19/anti-CD22) is linked to a toxinsuch as deglycosylated ricin A chain (e.g., Combotox).

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is a cytokine or is an agent that inducesincreased expression of a cytokine in the tumor microenvironment.Cytokines have important functions related to T cell expansion,differentiation, survival, and homeostasis. Cytokines that can beadministered to the subject receiving the combination therapy in theprovided methods or uses, recombinant receptors, cells and/orcompositions provided herein include one or more of IL-2, IL-4, IL-7,IL-9, IL-15, IL-18, and IL-21. In some embodiments, the cytokineadministered is IL-7, IL-15, or IL-21, or a combination thereof. In someembodiments, administration of the cytokine to the subject that hassub-optimal response to the administration of the engineered cells,e.g., CAR-expressing cells improves efficacy and/or anti-tumor activityof the administered cells, e.g., CAR-expressing cells.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is a cytokine, such as a protein that acton another cell as intercellular mediators. Examples of such cytokinesare lymphokines, monokines, and traditional polypeptide hormones.Included among the cytokines are growth hormones such as human growthhormone, N-methionyl human growth hormone, and bovine growth hormone;parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; fibroblast growth factor; prolactin; placentallactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-beta; platelet-growth factor;transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-alpha, beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as IL-1, IL-lalpha, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumornecrosis factor such as TNF-alpha or TNF-beta; and other polypeptidefactors including LIF and kit ligand (KL). As used herein, the termcytokine includes proteins from natural sources or from recombinant cellculture, and biologically active equivalents of the native sequencecytokines. For example, the immunomodulatory agent is a cytokine and thecytokine is IL-4, TNF-α, GM-CSF or IL-2.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, includes an interleukin-15 (IL-15)polypeptide, an interleukin-15 receptor alpha (IL-15Rα) polypeptide, orcombination thereof, e.g., hetIL-15 (Admune Therapeutics, LLC). hetIL-15is a heterodimeric non-covalent complex of IL-15 and IL-15Rα. hetIL-15is described in, e.g., U.S. Pat. No. 8,124,084, U.S. 2012/0177598, U.S.2009/0082299, U.S. 2012/0141413, and U.S. 2011/0081311. In someembodiments, the immunomodulatory agent can contain one or morecytokines. For example, the interleukin can include leukocyteinterleukin injection (Multikine), which is a combination of naturalcytokines.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is a modulator of adenosine levels and/oran adenosine pathway component. Adenosine can function as animmunomodulatory agent in the body. For example, adenosine and someadenosine analogs that non-selectively activate adenosine receptorsubtypes decrease neutrophil production of inflammatory oxidativeproducts (Cronstein et al., Ann. N.Y. Acad. Sci. 451:291, 1985; Robertset al., Biochem. J., 227:669, 1985; Schrier et al., J. Immunol.137:3284, 1986; Cronstein et al., Clinical Immunol. Immunopath. 42:76,1987). In some cases, concentration of extracellular adenosine oradenosine analogs can increase in specific environments, e.g., tumormicroenvironment (TME). In some cases, adenosine or adenosine analogsignaling depends on hypoxia or factors involved in hypoxia or itsregulation, e.g., hypoxia inducible factor (HIF). In some embodiments,increase in adenosine signaling can increase in intracellular cAMP andcAMP-dependent protein kinase that results in inhibition ofproinflammatory cytokine production, and can lead to the synthesis ofimmunosuppressive molecules and development of Tregs (Sitkovsky et al.,Cancer Immunol Res (2014) 2(7):598-605). In some embodiments, theadditional agent can reduce or reverse immunosuppressive effects ofadenosine, adenosine analogs and/or adenosine signaling. In someembodiments, the additional agent can reduce or reverse hypoxia-drivenA2-adenosinergic T cell immunosuppression. In some embodiments, theadditional agent is selected from among antagonists of adenosinereceptors, extracellular adenosine-degrading agents, inhibitors ofadenosine generation by CD39/CD73 ectoenzymes, and inhibitors ofhypoxia-HIF-1α signaling. In some embodiments, the additional agent isan adenosine receptor antagonist or agonist.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that inhibits the activityand/or an amount of an adenosine receptor. Particular embodimentscontemplate that inhibition or reduction of extracellular adenosine orthe adenosine receptor by virtue of an inhibitor of extracellularadenosine (such as an agent that prevents the formation of, degrades,renders inactive, and/or decreases extracellular adenosine), and/or anadenosine receptor inhibitor (such as an adenosine receptor antagonist)can enhance immune response, such as a macrophage, neutrophil,granulocyte, dendritic cell, T- and/or B cell-mediated response. Inaddition, inhibitors of the Gs protein mediated cAMP dependentintracellular pathway and inhibitors of the adenosine receptor-triggeredGi protein mediated intracellular pathways, can also increase acute andchronic inflammation.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an adenosine receptor antagonist oragonist, e.g., an antagonist or agonist of one or more of the adenosinereceptors A2a, A2b, A1, and A3. A1 and A3 inhibit, and A2a and A2bstimulate, respectively, adenylate cyclase activity. Certain adenosinereceptors, such as A2a, A2b, and A3, can suppress or reduce the immuneresponse during inflammation. Thus, antagonizing immunosuppressiveadenosine receptors can augment, boost or enhance immune response, e.g.,immune response from administered cells, e.g., CAR-expressing T cells.In some embodiments, the additional agent inhibits the production ofextracellular adenosine and adenosine-triggered signaling throughadenosine receptors. For example, enhancement of an immune response,local tissue inflammation, and targeted tissue destruction can beenhanced by inhibiting or reducing the adenosine-producing local tissuehypoxia; by degrading (or rendering inactive) accumulated extracellularadenosine; by preventing or decreasing expression of adenosine receptorson immune cells; and/or by inhibiting/antagonizing signaling byadenosine ligands through adenosine receptors.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an adenosine receptor antagonist. Insome embodiments, the antagonist is small molecule or chemical compoundof an adenosine receptor, such as the A2a, A2b, or A3 receptor. In someembodiments, the antagonist is a peptide, or a pepidomimetic, that bindsthe adenosine receptor but does not trigger a Gi protein dependentintracellular pathway. Examples of such antagonists are described inU.S. Pat. Nos. 5,565,566; 5,545,627, 5,981,524; 5,861,405; 6,066,642;6,326,390; 5,670,501; 6,117,998; 6,232,297; 5,786,360; 5,424,297;6,313,131, 5,504,090; and 6,322,771.

In some embodiments, the additional agent is an A2 receptor (A2R)antagonist, such as an A2a antagonist. Exemplary A2R antagonistsinclude, but are not limited to, KW6002 (istradefyline), SCH58261,caffeine, paraxanthine, 3,7-dimethyl-1-propargylxanthine (DMPX),8-(m-chlorostyryl) caffeine (CSC), MSX-2, MSX-3, MSX-4, CGS-15943,ZM-241385, SCH-442416, preladenant, vipadenant (BII014), V2006, ST-1535,SYN-115, PSB-1115, ZM241365, FSPTP, and an inhibitory nucleic acidtargeting A2R expression, e.g., siRNA or shRNA, or any antibodies orantigen-binding fragment thereof that targets an A2R. In someembodiments, the additional agent is an A2R antagonist described in,e.g., Ohta et al., Proc Natl Acad Sci USA (2006) 103:13132-13137; Jin etal., Cancer Res. (2010) 70(6):2245-2255; Leone et al., Computational andStructural Biotechnology Journal (2015) 13:265-272; Beavis et al., ProcNatl Acad Sci USA (2013) 110:14711-14716; and Pinna, A., Expert OpinInvestig Drugs (2009) 18:1619-1631; Sitkovsky et al., Cancer Immunol Res(2014) 2(7):598-605; U.S. Pat. Nos. 8,080,554; 8,716,301; US20140056922; WO2008/147482; U.S. Pat. No. 8,883,500; US 20140377240;WO02/055083; U.S. Pat. Nos. 7,141,575; 7,405,219; 8,883,500; 8,450,329and 8,987,279).

In particular embodiments, an adenosine receptor antagonist that is anantisense molecule, inhibitory nucleic acid molecule (e.g., smallinhibitory RNA (siRNA)) or catalytic nucleic acid molecule (e.g. aribozyme) that specifically binds mRNA encoding an adenosine receptor.In some embodiments, the antisense molecule, inhibitory nucleic acidmolecule or catalytic nucleic acid molecule binds nucleic acids encodingA2a, A2b, or A3. In some embodiments, an antisense molecule, inhibitorynucleic acid molecule or catalytic nucleic acid targets biochemicalpathways downstream of the adenosine receptor. For example, theantisense molecule or catalytic nucleic acid can inhibit an enzymeinvolved in the Gs protein- or Gi protein-dependent intracellularpathway. In some embodiments, the additional agent includes dominantnegative mutant form of an adenosine receptor, such as A2a, A2b, or A3.

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is an agent that inhibits extracellularadenosine. Agents that inhibit extracellular adenosine include agentsthat render extracellular adenosine non-functional (or decrease suchfunction), such as a substance that modifies the structure of adenosineto inhibit the ability of adenosine to signal through adenosinereceptors. In some embodiments, the additional agent is an extracellularadenosine-generating or adenosine-degrading enzyme, a modified formthereof or a modulator thereof. For example, in some embodiments, theadditional agent is an enzyme (e.g. adenosine deaminase) or anothercatalytic molecule that selectively binds and destroys the adenosine,thereby abolishing or significantly decreasing the ability ofendogenously formed adenosine to signal through adenosine receptors andterminate inflammation.

In some embodiments, the additional agent is an adenosine deaminase(ADA) or a modified form thereof, e.g., recombinant ADA and/orpolyethylene glycol-modified ADA (ADA-PEG), which can inhibit localtissue accumulation of extracellular adenosine. ADA-PEG has been used intreatment of patients with ADA SCID (Hershfield (1995) Hum Mutat.5:107). In some embodiments, an agent that inhibits extracellularadenosine includes agents that prevent or decrease formation ofextracellular adenosine, and/or prevent or decrease the accumulation ofextracellular adenosine, thereby abolishing, or substantiallydecreasing, the immunosuppressive effects of adenosine. In someembodiments, the additional agent specifically inhibits enzymes andproteins that are involved in regulation of synthesis and/or secretionof pro-inflammatory molecules, including modulators of nucleartranscription factors. Suppression of adenosine receptor expression orexpression of the Gs protein- or Gi protein-dependent intracellularpathway, or the cAMP dependent intracellular pathway, can result in anincrease/enhancement of immune response.

In some embodiments, the additional agent can target ectoenzymes thatgenerate or produce extracellular adenosine. In some embodiments, theadditional agent targets CD39 and CD73 ectoenzymes, which function intandem to generate extracellular adenosine. CD39 (also calledectonucleoside triphosphate diphosphohydrolase) converts extracellularATP (or ADP) to 5′AMP. Subsequently, CD73 (also called 5′ nucleotidase)converts 5′ AMP to adenosine. The activity of CD39 is reversible by theactions of NDP kinase and adenylate kinase, whereas the activity of CD73is irreversible. CD39 and CD73 are expressed on tumor stromal cells,including endothelial cells and Tregs, and also on many cancer cells.For example, the expression of CD39 and CD73 on endothelial cells isincreased under the hypoxic conditions of the tumor microenvironment.Tumor hypoxia can result from inadequate blood supply and disorganizedtumor vasculature, impairing delivery of oxygen (Carroll and Ashcroft(2005), Expert. Rev. Mol. Med. 7(6):1-16). Hypoxia also inhibitsadenylate kinase (AK), which converts adenosine to AMP, leading to veryhigh extracellular adenosine concentration. Thus, adenosine is releasedat high concentrations in response to hypoxia, which is a condition thatfrequently occurs the tumor microenvironment (TME), in or around solidtumors. In some embodiments, the additional agent is one or more ofanti-CD39 antibody or antigen binding fragment thereof, anti-CD73antibody or antigen binding fragment thereof, e.g., MEDI9447 or TY/23,α-β-methylene-adenosine diphosphate (ADP), ARL 67156, POM-3, IPH52 (see,e.g., Allard et al. Clin Cancer Res (2013) 19(20):5626-5635; Hausler etal., Am J Transl Res (2014) 6(2):129-139; Zhang, B., Cancer Res. (2010)70(16):6407-6411).

In some embodiments, the additional agent that is administered in accordwith the provided methods, and/or with the provided articles ofmanufacture or compositions, is a chemotherapeutic agent (sometimesreferred to as a cytotoxic agent). In particular embodiments, thechemotherapeutic agent is any agent known to those of skill in the artto be effective for the treatment, prevention or amelioration ofhyperproliferative disorders such as cancer. Chemotherapeutic agentsinclude, but are not limited to, small molecules, synthetic drugs,peptides, polypeptides, proteins, nucleic acids (e.g., DNA and RNApolynucleotides including, but not limited to, antisense nucleotidesequences, triple helices and nucleotide sequences encoding biologicallyactive proteins, polypeptides or peptides), antibodies, synthetic ornatural inorganic molecules, mimetic agents, and synthetic or naturalorganic molecules. In particular embodiments, chemotherapeutic drugsinclude alkylating agents, anthracyclines, cytoskeletal disruptors(taxanes), epothilones, histone deacetylase inhibitors, topoisomeraseinhibitors, topoisomerase II inhibitors, kinase inhibitors, nucleotideanalogs and precursor analogs, peptide antibiotics, platinum-basedagents, and vinca alkaloids and derivatives.

Chemotherapeutic agents may include, but are not limited to, abarelix,aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine,amifostine, anastrozole, arsenic trioxide, asparaginase, BCG live,bevaceizumab, bexarotene, bleomycin, bortezomib, busulfan, calusterone,camptothecin, capecitabine, carboplatin, carmustine, celecoxib,cetuximab, chlorambucil, cinacalcet, cisplatin, cladribine,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, darbepoetinalfa, daunorubicin, denileukin diftitox, dexrazoxane, docetaxel,doxorubicin, dromostanolone, Elliott's B solution, epirubicin, epoetinalfa, estramustine, etoposide, exemestane, filgrastim, floxuridine,fludarabine, fluorouracil, fulvestrant, gemcitabine, gemtuzumabozogamicin, gefitinib, goserelin, hydroxyurea, ibritumomab tiuxetan,idarubicin, ifosfamide, imatinib, interferon alfa-2a, interferonalfa-2b, irinotecan, letrozole, leucovorin, levamisole, lomustine,meclorethamine, megestrol, melphalan, mercaptopurine, mesna,methotrexate, methoxsalen, methylprednisolone, mitomycin C, mitotane,mitoxantrone, nandrolone, nofetumomab, oblimersen, oprelvekin,oxaliplatin, paclitaxel, pamidronate, pegademase, pegaspargase,pegfilgrastim, pemetrexed, pentostatin, pipobroman, plicamycin,polifeprosan, porfimer, procarbazine, quinacrine, rasburicase,rituximab, sargramostim, streptozocin, talc, tamoxifen, tarceva,temozolomide, teniposide, testolactone, thioguanine, thiotepa,topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracilmustard, valrubicin, vinblastine, vincristine, vinorelbine, andzoledronate.

In some embodiments, the additional agent is an inhibitor of hypoxiainducible factor 1 alpha (HIF-1α) signaling. Exemplary inhibitors ofHIF-1α include digoxin, acriflavine, sirtuin-7 and ganetespib.

In some embodiments, the additional agent includes a protein tyrosinephosphatase inhibitor, e.g., a protein tyrosine phosphatase inhibitordescribed herein. In some embodiments, the protein tyrosine phosphataseinhibitor is an SHP-1 inhibitor, e.g., an SHP-1 inhibitor describedherein, such as, e.g., sodium stibogluconate. In some embodiments, theprotein tyrosine phosphatase inhibitor is an SHP-2 inhibitor, e.g., anSHP-2 inhibitor described herein.

In some embodiments, the additional agent is a kinase inhibitor. Kinaseinhibitors, such as a CDK4 kinase inhibitor, a BTK kinase inhibitor, aMNK kinase inhibitor, or a DGK kinase inhibitor, can regulate theconstitutively active survival pathways that exist in tumor cells and/ormodulate the function of immune cells. In some embodiments, the kinaseinhibitor is a Bruton's tyrosine kinase (BTK) inhibitor, e.g.,ibrutinib. In some embodiments, the kinase inhibitor is aphosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor. In someembodiments, the kinase inhibitor is a CDK4 inhibitor, e.g., a CDK4/6inhibitor. In some embodiments, the kinase inhibitor is an mTORinhibitor, such as, e.g., rapamycin, a rapamycin analog, OSI-027. ThemTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor. In someembodiments, the kinase inhibitor is an MNK inhibitor, or a dualPI3K/mTOR inhibitor. In some embodiments, other exemplary kinaseinhibitors include the AKT inhibitor perifosine, the mTOR inhibitortemsirolimus, the Src kinase inhibitors dasatinib and fostamatinib, theJAK2 inhibitors pacritinib and ruxolitinib, the PKCβ inhibitorsenzastaurin and bryostatin, and the AAK inhibitor alisertib.

In some embodiments, the kinase inhibitor is a BTK inhibitor selectedfrom ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764;HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In some embodiments,the BTK inhibitor does not reduce or inhibit the kinase activity ofinterleukin-2-inducible kinase (ITK), and is selected from GDC-0834;RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; andLFM-A13.

In some embodiments, the kinase inhibitor is a BTK inhibitor, e.g.,ibrutinib(1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one;also known as PCI-32765). In some embodiments, the kinase inhibitor is aBTK inhibitor, e.g., ibrutinib (PCI-32765), and the ibrutinib isadministered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg,440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg(e.g., 250 mg, 420 mg or 560 mg) daily for a period of time, e.g., dailyfor 21 day cycle, or daily for 28 day cycle. In some embodiments, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib areadministered. In some embodiments, the BTK inhibitor is a BTK inhibitordescribed in International Application WO 2015/079417.

In some embodiments, the kinase inhibitor is a PI3K inhibitor. PI3K iscentral to the PI3K/Akt/mTOR pathway involved in cell cycle regulationand lymphoma survival. Exemplary PI3K inhibitor includes idelalisib(PI3Kδ inhibitor). In some embodiments, the additional agent isidelalisib and rituximab.

In some embodiments, the additional agent is an inhibitor of mammaliantarget of rapamycin (mTOR). In some embodiments, the kinase inhibitor isan mTOR inhibitor selected from temsirolimus; ridaforolimus (also knownas AP23573 and MK8669); everolimus (RAD001); rapamycin (AY22989);simapimod; AZD8055; PF04691502; SF1126; and XL765. In some embodiments,the additional agent is an inhibitor of mitogen-activated protein kinase(MAPK), such as vemurafenib, dabrafenib, and trametinib.

In some embodiments, the additional agent is an agent that regulatespro- or anti-apoptotic proteins. In some embodiments, the additionalagent includes a B-cell lymphoma 2 (BCL-2) inhibitor (e.g., venetoclax,also called ABT-199 or GDC-0199; or ABT-737). Venetoclax is a smallmolecule(4-(4-{[2-(4-Chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) that inhibits the anti-apoptotic protein, BCL-2.Other agents that modulate pro- or anti-apoptotic protein include BCL-2inhibitor ABT-737, navitoclax (ABT-263); Mcl-1 siRNA or Mcl-1 inhibitorretinoid N-(4-hydroxyphenyl) retinamide (4-HPR) for maximal efficacy. Insome embodiments, the additional agent provides a pro-apoptotic stimuli,such as recombinant tumor necrosis factor-related apoptosis-inducingligand (TRAIL), which can activate the apoptosis pathway by binding toTRAIL death receptors DR-4 and DR-5 on tumor cell surface, or TRAIL-R2agonistic antibodies.

In some embodiments, the additional agent includes a cytotoxic agent,e.g., CPX-351 (Celator Pharmaceuticals), cytarabine, daunorubicin,vosaroxin (Sunesis Pharmaceuticals), sapacitabine (CyclacelPharmaceuticals), idarubicin, or mitoxantrone. In some embodiments, theadditional agent includes a hypomethylating agent, e.g., a DNAmethyltransferase inhibitor, e.g., azacitidine or decitabine.

In another embodiment, the additional therapy is a transplantation,e.g., allogeneic stem cell transplant.

In some embodiments, the additional therapy is a lymphodepletingtherapy. In some embodiments, lymphodepletion is performed on a subject,e.g., prior to administering engineered cells, e.g., CAR-expressingcells. In some embodiments, the lymphodepletion comprises administeringone or more of melphalan, Cytoxan, cyclophosphamide, and fludarabine. Insome embodiments, a lymphodepleting chemotherapy is administered to thesubject prior to, concurrently with, or after administration (e.g.,infusion) of engineered cells, e.g., CAR-expressing cells. In anexample, the lymphodepleting chemotherapy is administered to the subjectprior to administration of engineered cells, e.g., CAR-expressing cells.

In some embodiments, the additional agent is an oncolytic virus. In someembodiments, oncolytic viruses are capable of selectively replicating inand triggering the death of or slowing the growth of a cancer cell. Insome cases, oncolytic viruses have no effect or a minimal effect onnon-cancer cells. An oncolytic virus includes but is not limited to anoncolytic adenovirus, oncolytic Herpes Simplex Viruses, oncolyticretrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolyticSinbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g.,oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolyticmeasles virus, or oncolytic vesicular stomatitis virus (VSV)).

Other exemplary combination therapy, treatment and/or agents includeanti-allergenic agents, anti-emetics, analgesics and adjunct therapies.In some embodiments, the additional agent includes cytoprotectiveagents, such as neuroprotectants, free-radical scavengers,cardioprotectors, anthracycline extravasation neutralizers andnutrients.

In some embodiments, an antibody used as an additional agent isconjugated or otherwise bound to a therapeutic agent, e.g., achemotherapeutic agent (e.g., Cytoxan, fludarabine, histone deacetylaseinhibitor, demethylating agent, peptide vaccine, anti-tumor antibiotic,tyrosine kinase inhibitor, alkylating agent, anti-microtubule oranti-mitotic agent), anti-allergic agent, anti-nausea agent (oranti-emetic), pain reliever, or cytoprotective agent described herein.In some embodiments, the additional agent is an antibody-drug conjugate.

Any of the additional agents described herein can be prepared andadministered as a combination therapy described in the provided methods,uses, articles of manufacture or compositions, such as in pharmaceuticalcompositions comprising one or more agents of the combination therapyand a pharmaceutically acceptable carrier, such as any described herein.In some embodiments, the combination therapy in the provided methods,uses, articles of manufacture or compositions can be administeredsimultaneously, concurrently or sequentially, in any order with theadditional agents, therapy or treatment, wherein such administrationprovides therapeutically effective levels each of the agents in the bodyof the subject. In some embodiments, the additional agent can beco-administered with the combination therapy in the provided methods,uses, articles of manufacture or compositions, for example, as part ofthe same pharmaceutical composition or using the same method ofdelivery. In some embodiments, the additional agent is administeredsimultaneously with the cell therapy, e.g. dose of engineered T cells(e.g. CAR⁺ T cells), but in separate compositions. In some embodiments,the additional agent is incubated with the engineered cell, e.g.,CAR-expressing cells, prior to administration of the cells.

In some examples, the one or more additional agents are administeredsubsequent to or prior to the administration of the cell therapy, e.g.dose of engineered T cells (e.g. CAR⁺ T cells), separated by a selectedtime period. In some examples, the time period is 1 day, 2 days, 3 days,4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, or3 months. In some examples, the one or more additional agents areadministered multiple times. In some embodiments, the additional agentis administered prior to the cell therapy, e.g. dose of engineered Tcells (CAR⁺ T cells) in the provided methods, uses, articles ofmanufacture or compositions, e.g., two weeks, 12 days, 10 days, 8 days,one week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day before theadministration. In some embodiments, the additional agent isadministered after the cell therapy, e.g. dose of engineered T cells(e.g. CAR⁺ T cells) in the provided methods, uses, articles ofmanufacture or compositions, e.g., two weeks, 12 days, 10 days, 8 days,one week, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day after theadministration.

The dose of the additional agent can be any therapeutically effectiveamount, e.g., any dose amount described herein, and the appropriatedosage of the additional agent may depend on the type of disease to betreated, the type, dose and/or frequency of the binding molecule,recombinant receptor, cell and/or composition administered, the severityand course of the disease, previous therapy, the patient's clinicalhistory and response to cell therapy, e.g. dose of engineered T cells(CAR⁺ T cells), and the discretion of the attending physician.

VI. Articles of Manufacture and Kits

Also provided are articles of manufacture and kits containing engineeredcells expressing a recombinant receptor or compositions thereof, andoptionally instructions for use, for example, instructions foradministering, according to the provided methods.

In some embodiments, provided are articles of manufacture and/or kitsthat include a composition comprising a therapeutically effective amountof any of the engineered cells described herein, and instructions foradministering, to a subject for treating a disease or condition. In someembodiments, the instructions can specify some or all of the elements ofthe methods provided herein. In some embodiments, the instructionsspecify particular instructions for administration of the cells for celltherapy, e.g., doses, timing, selection and/or identification ofsubjects for administration and conditions for administration. In someembodiments, the articles of manufacture and/or kits further include oneor more additional agents for therapy, e.g., lymphodepleting therapyand/or combination therapy, such as any described herein and optionallyfurther includes instructions for administering the additional agent fortherapy. In some embodiments, the articles of manufacture and/or kitsfurther comprise an agent for lymphodepleting therapy, and optionallyfurther includes instructions for administering the lymphodepletingtherapy. In some embodiments, the instructions can be included as alabel or package insert accompanying the compositions foradministration.

In some embodiments, the instructions specify the criteria for selectionor identification of subjects for therapy. In some embodiments, suchcriteria include subjects having NHL or sub-type thereof and/or ahigh-risk NHL. In some embodiments, the instructions specify that thesubjects to be treated include subjects having a disease or conditioncharacterized or determined to be aggressive NHL, diffuse large B celllymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), Tcell/histocyte-rich large B cell lymphoma (TCHRBCL), Burkitt's lymphoma,mantle cell lymphoma (MCL), and/or follicular lymphoma (FL). Inparticular embodiments, the subject to be treated include subjects withaggressive NHL, in particular, with diffuse large B-cell lymphoma(DLBCL), not otherwise specified (NOS) and in some aspects including denovo and transformed from indolent). In some aspects, the subject orpopulation to be treated may include and/or further include subjectswith primary mediastinal B-cell lymphoma (PMBCL) or follicular lymphomagrade 3B (FL3B). In some embodiments, the subject or population to betreated include those subjects having poor performance status. In someaspects, the population to be treated includes, e.g., subjects having anEastern Cooperative Oncology Group Performance Status (ECOG) that isanywhere from 0-2. In other aspects of any of the embodiments, thesubjects to be treated include ECOG 0-1 or do not include ECOG2subjects. In some embodiments, of any of the embodiments, the subjectsto be treated have failed two or more prior therapies. In someembodiments, the subject does not have DLBCL transformed from marginalzone lymphoma (MZL) and chronic lymphocytic leukemia (CLL; Richter's)and/or has a DLBCL characterized as de novo or transformed from anindolent disease. In some embodiments, the subject has mantle celllymphoma (MCL). In some embodiments, the instructions specify theadministration of the cell therapy is for a subject that is or has beenidentified as having a double/triple hit lymphoma (or high-grade B-celllymphoma, with MYC and BCL2 and/or BCL6 rearrangements with DLBCLhistology) has been identified as having a chemorefractory lymphoma,(e.g., chemorefractory DLBCL) and/or that has not achieved completeremission (CR) in response to a prior therapy.

In some embodiments, the instructions specify the dose of cells to beadministered. For example, in some embodiments, the dose specified inthe instructions include a total recombinant receptor (e.g.,CAR)-expressing cells between about 1×10⁶ and 3×10⁸, e.g., in the rangeof about 1×10⁷ to 2×10⁸ such cells, such as 1×10⁷, 5×10⁷, 1×10⁸ or1.5×10⁸total such cells, or the range between any two of the foregoingvalues. In some embodiments, the patient is administered multiple doses,and each of the doses or the total dose can be within any of theforegoing values.

In some embodiments, the article of manufacture or kit comprises acontainer, optionally a vial comprising a plurality of CD4⁺ T cellsexpressing a recombinant receptor, and a container, optionally a vialcomprising a plurality of CD8⁺ T cells expressing a recombinantreceptor. In some embodiments, the article of manufacture or kitcomprises a container, optionally a vial comprising a plurality of CD4⁺T cells expressing a recombinant receptor, and further comprises, in thesame container, a plurality of CD8⁺ T cells expressing a recombinantreceptor. In some embodiments, a cryoprotectant is included with thecells. In some aspects the container is a bag.

In some embodiments, the container such as the vial comprises greaterthan or greater than about 10×10⁶ T cells or recombinantreceptor-expressing T cells, greater than or greater than about 15×10⁶ Tcells or recombinant receptor-expressing T cells, greater than orgreater than about 25×10⁶ T cells or recombinant receptor-expressing Tcell. In some aspects, the vial comprises between about 10 million cellsper ml and about 70 million cells per ml, between about 10 million cellsper ml and about 50 million cells per ml, between about 10 million cellsper ml and about 25 million cells per ml, between about 10 million cellsper ml and about 15 million cells per ml, 15 million cells per ml andabout 70 million cells per ml, between about 15 million cells per ml andabout 50 million cells per ml, between about 15 million cells per ml andabout 25 million cells per ml, between about 25 million cells per ml andabout 70 million cells per ml, between about 25 million cells per ml andabout 50 million cells per ml, and between about 50 million cells per mland about 70 million cells per ml.

In some embodiments, the plurality of vials or plurality of cells orunit dose of cells specified for administration, collectively, comprisesa dose of cells comprising from or from about 1×10⁵ to 5×10⁸ totalrecombinant receptor-expressing T cells or total T cells, 1×10⁵ to 1×10⁸total recombinant receptor-expressing T cells or total T cells, from orfrom about 5×10⁵ to 1×10⁷ total recombinant receptor-expressing T cellsor total T cells, or from or from about 1×10⁶ to 1×10⁷ total recombinantreceptor-expressing T cells or total T cells, each inclusive. In someaspects, the article comprises one or more unit dose of the CD4⁺ andCD8⁺ cells or of the CD4⁺ receptor⁺ cells and CD8⁺receptor⁺ cells,wherein the unit dose comprises between at or about 1×10⁷ and at orabout 2×10⁸ recombinant receptor-expressing T cells, between at or about5×10⁷ and at or about 1.5×10⁸ recombinant receptor-expressing T cells,at or about 5×10⁷ recombinant receptor-expressing T cells, at or about1×10⁸ recombinant receptor-expressing T cells, or at or about 1.5×10⁸recombinant receptor-expressing T cells, optionally wherein theinformation in the article specifies administration of one or of aplurality of unit doses and/or a volume corresponding to such one orplurality of unit doses. In some cases, the article comprises one ormore unit doses of the CD8⁺ cells, wherein the dose comprises between ator about 5×10⁶ and at or about 1×10⁸ recombinant receptor-expressingCD8⁺ T cells, the dose comprises between at or about 1×10⁷ and at orabout 0.75×10⁸ recombinant receptor-expressing CD8⁺ T cells, the dosecomprises at or about 2.5×10⁷ recombinant receptor-expressing CD8⁺ Tcells, or the dose comprises at or about 5×10⁷ recombinantreceptor-expressing CD8⁺ T cells, or the dose comprises at or about0.75×10⁸ recombinant receptor-expressing CD8⁺ T cells, optionallywherein the information in the article specifies administration of oneor of a plurality of unit doses and/or a volume corresponding to suchone or plurality of unit doses. In some embodiments, the cells in thearticle, collectively, comprise a dose of cells comprising no more than1×10⁸ total recombinant receptor-expressing T cells or total T cells, nomore than 1×10⁷ total recombinant receptor-expressing T cells or total Tcells, no more than 0.5×10⁷ total recombinant receptor-expressing Tcells or total T cells, no more than 1×10⁶ total recombinantreceptor-expressing T cells or total T cells, no more than 0.5×10⁶ totalrecombinant receptor-expressing T cells or total T cells.

In some embodiments, each vial or the plurality of vials or plurality ofcells or unit dose of cells specified for administration, collectively,comprises a flat dose of cells or fixed dose of cells such that the doseof cells is not tied to or based on the body surface area or weight of asubject.

In some embodiments, a unit dose of a cell is or comprises the number oramount of cells, such as engineered T cells, that can be administered toa subject or a patient in a single dose.

In some embodiments, each vial or the plurality of vials or plurality ofcells or unit dose of cells specified for administration, collectively,comprises a dose that includes fewer than about 5×10⁸ total recombinantreceptor (e.g., CAR)-expressing cells, T cells, or peripheral bloodmononuclear cells (PBMCs), e.g., in the range of about 1×10⁶ to 5×10⁸such cells, such as 2×10⁶, 5×10⁶, 1×10⁷, 5×10⁷, 1×10⁸, or 5×10⁸ totalsuch cells, or the range between any two of the foregoing values.

In some embodiments, each vial or the plurality of vials or plurality ofcells or unit dose of cells specified for administration, collectively,comprises a dose of genetically engineered cells comprising from or fromabout 1×10⁵ to 5×10⁸ total CAR-expressing T cells, 1×10⁵ to 2.5×10⁸total CAR-expressing T cells, 1×10⁵ to 1×10⁸ total CAR-expressing Tcells, 1×10⁵ to 5×10⁷ total CAR-expressing T cells, 1×10⁵ to 2.5×10⁷total CAR-expressing T cells, 1×10⁵ to 1×10⁷ total CAR-expressing Tcells, 1×10⁵ to 5×10⁶ total CAR-expressing T cells, 1×10⁵ to 2.5×10⁶total CAR-expressing T cells, 1×10⁵ to 1×10⁶ total CAR-expressing Tcells, 1×10⁶ to 5×10⁸ total CAR-expressing T cells, 1×10⁶ to 2.5×10⁸total CAR-expressing T cells, 1×10⁶ to 1×10⁸ total CAR-expressing Tcells, 1×10⁶ to 5×10⁷ total CAR-expressing T cells, 1×10⁶ to 2.5×10⁷total CAR-expressing T cells, 1×10⁶ to 1×10⁷ total CAR-expressing Tcells, 1×10⁶ to 5×10⁶ total CAR-expressing T cells, 1×10⁶ to 2.5×10⁶total CAR-expressing T cells, 2.5×10⁶ to 5×10⁸ total CAR-expressing Tcells, 2.5×10⁶ to 2.5×10⁸ total CAR-expressing T cells, 2.5×10⁶ to 1×10⁸total CAR-expressing T cells, 2.5×10⁶ to 5×10⁷ total CAR-expressing Tcells, 2.5×10⁶ to 2.5×10⁷ total CAR-expressing T cells, 2.5×10⁶ to 1×10⁷total CAR-expressing T cells, 2.5×10⁶ to 5×10⁶ total CAR-expressing Tcells, 5×10⁶ to 5×10⁸ total CAR-expressing T cells, 5×10⁶ to 2.5×10⁸total CAR-expressing T cells, 5×10⁶ to 1×10⁸ total CAR-expressing Tcells, 5×10⁶ to 5×10⁷ total CAR-expressing T cells, 5×10⁶ to 2.5×10⁷total CAR-expressing T cells, 5×10⁶ to 1×10⁷ total CAR-expressing Tcells, 1×10⁷ to 5×10⁸ total CAR-expressing T cells, 1×10⁷ to 2.5×10⁸total CAR-expressing T cells, 1×10⁷ to 1×10⁸ total CAR-expressing Tcells, 1×10⁷ to 5×10⁷ total CAR-expressing T cells, 1×10⁷ to 2.5×10⁷total CAR-expressing T cells, 2.5×10⁷ to 5×10⁸ total CAR-expressing Tcells, 2.5×10⁷ to 2.5×10⁸ total CAR-expressing T cells, 2.5×10⁷ to 1×10⁸total CAR-expressing T cells, 2.5×10⁷ to 5×10⁷ total CAR-expressing Tcells, 5×10⁷ to 5×10⁸ total CAR-expressing T cells, 5×10⁷ to 2.5×10⁸total CAR-expressing T cells, 5×10⁷ to 1×10⁸ total CAR-expressing Tcells, 1×10⁸ to 5×10⁸ total CAR-expressing T cells, 1×10⁸ to 2.5×10⁸total CAR-expressing T cells, or 2.5×10⁸ to 5×10⁸ total CAR-expressing Tcells.

In some embodiments, each vial or the plurality of vials or plurality ofcells or unit dose of cells specified for administration, collectively,comprises a dose of genetically engineered cells comprising at least orat least about 1×10⁵ CAR-expressing cells, at least or at least about2.5×10⁵ CAR-expressing cells, at least or at least about 5×10⁵CAR-expressing cells, at least or at least about 1×10⁶ CAR-expressingcells, at least or at least about 2.5×10⁶ CAR-expressing cells, at leastor at least about 5×10⁶ CAR-expressing cells, at least or at least about1×10⁷ CAR-expressing cells, at least or at least about 2.5×10⁷CAR-expressing cells, at least or at least about 5×10⁷ CAR-expressingcells, at least or at least about 1×10⁸ CAR-expressing cells, at leastor at least about 2.5×10⁸ CAR-expressing cells, or at least or at leastabout 5×10⁸ CAR-expressing cells.

In some embodiments, the instructions for administration of a dose ofengineered cell specify administering a number of cell from or fromabout 1×10⁵ to 5×10⁸ total recombinant receptor-expressing cells, totalT cells, or total peripheral blood mononuclear cells (PBMCs), from orfrom about 5×10⁵ to 1×10⁷ total recombinant receptor-expressing cells,total T cells, or total peripheral blood mononuclear cells (PBMCs) orfrom or from about 1×10⁶ to 1×10⁷ total recombinant receptor-expressingcells, total T cells, or total peripheral blood mononuclear cells(PBMCs), each inclusive. In some embodiments, the cell therapy comprisesadministration of a dose of cells comprising a number of cells at leastor at least about 1×10⁵ total recombinant receptor-expressing cells,total T cells, or total peripheral blood mononuclear cells (PBMCs), suchat least or at least 1×10⁶, at least or at least about 1×10⁷, at leastor at least about 1×10⁸ of such cells. In some embodiments, the numberis with reference to the total number of CD3+ or CD8+, in some casesalso recombinant receptor-expressing (e.g. CAR+) cells. In someembodiments, the cell therapy comprises administration of a dosecomprising a number of cell from or from about 1×10⁵ to 5×10⁸ CD3+ orCD8+ total T cells or CD3+ or CD8+ recombinant receptor-expressingcells, from or from about 5×10⁵ to 1×10⁷ CD3+ or CD8+ total T cells orCD3+ or CD8+ recombinant receptor-expressing cells, or from or fromabout 1×10⁶ to 1×10⁷ CD3+ or CD8+ total T cells or CD3+ or CD8+recombinant receptor-expressing cells, each inclusive. In someembodiments, the cell therapy comprises administration of a dosecomprising a number of cell from or from about 1×10⁵ to 5×10⁸ totalCD3+/CAR+ or CD8+/CAR+ cells, from or from about 5×10⁵ to 1×10⁷ totalCD3+/CAR+ or CD8+/CAR+ cells, or from or from about 1×10⁶ to 1×10⁷ totalCD3+/CAR+ or CD8+/CAR+ cells, each inclusive.

In some embodiments, the instructions can specify dosage regimen andtiming of the administration. For example, in some embodiments, theinstructions can specify administering to the subject multiple doses,e.g., two or more doses, of the cells. In some embodiments, theinstructions specify the timing of the multiple doses, e.g., the seconddose being administered approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose; and/or thedosage amount in each dose.

In some embodiments, the article of manufacture or kit comprises aplurality of CD4⁺ T cells expressing a recombinant receptor, andinstructions for administering, to a subject having a disease orcondition, all or a portion of the plurality of CD4⁺ T cells and furtheradministering CD8⁺ T cells expressing a recombinant receptor. In someembodiments, the instructions specify administering the CD4⁺ T cellsprior to administering the CD8⁺ cells. In some cases, the instructionsspecify administering the CD8⁺ T cells prior to administering the CD4⁺cells. In some embodiments, the article of manufacture or kit comprisesa plurality of CD8⁺ T cells expressing a recombinant receptor, andinstructions for administering, to a subject having a disease orcondition, all or a portion of the plurality of CD8⁺ T cells and CD4⁺ Tcells expressing a recombinant receptor. In some embodiments, theinstructions specify dosage regimen and timing of the administration ofthe cells.

In some aspects, the instructions specify administering all or a portionof the CD4⁺ T cells and the all or a portion of the CD8⁺ T cells 0 to 12hours apart, 0 to 6 hours apart or 0 to 2 hours apart. In some cases,the instructions specify administering the CD4⁺ T cells and the CD8⁺ Tcells no more than 2 hours, no more than 1 hour, no more than 30minutes, no more than 15 minutes, no more than 10 minutes or no morethan 5 minutes apart.

In some embodiments, the instructions specify the dose or number ofcells or cell type(s) and/or a ratio of cell types, e.g., individualpopulations or sub-types, such as the CD4⁺ to CD8⁺ ratio. In someembodiments, the populations or sub-types of cells, such as CD8⁺ andCD4⁺ T cells. For example, in some embodiments, the instructions specifythat the cells are administered at or within a tolerated range of anoutput ratio of multiple cell populations or sub-types, such as CD4⁺ andCD8⁺ cells or sub-types, of between at or about 5:1 and at or about 5:1(or greater than about 1:5 and less than about 5:1), or between at orabout 1:3 and at or about 3:1 (or greater than about 1:3 and less thanabout 3:1), such as between at or about 2:1 and at or about 1:5 (orgreater than about 1:5 and less than about 2:1, such as at or about 5:1,4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1,1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5,1:1.6, 1:1.7, 1:1.8, 1:1.9:1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.In some aspects, the tolerated difference is within about 1%, about 2%,about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%,about 30%, about 35%, about 40%, about 45%, about 50% of the desiredratio, including any value in between these ranges.

In some embodiments, the articles of manufacture and/or kits furtherinclude one or more additional agents for therapy, e.g., lymphodepletingtherapy and/or combination therapy, as described herein, and optionallyinstructions for administering the additional agents. In some examples,the articles of manufacture may further contain one or more therapeuticagents. In some embodiments, the therapeutic agent is animmunomodulatory agent, a cytotoxic agent, an anti-cancer agent or aradiotherapeutic.

In some embodiments, the articles of manufacture and/or kits furtherinclude one or more agents or treatments for treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity and/or instructions for the administration of one or moreagents or treatments for treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity in thesubject. In some embodiments, the agent is or comprises an anti-IL-6antibody or anti-IL-6 receptor antibody. For example, in someembodiments, the agent or treatment is or comprises an agent selectedfrom among tocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab(CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136),CPSI-2634, ARGX-109, FE301 and FM101. For example, in some embodiments,the agent or treatment is or comprises one or more of a steroid; anantagonist or inhibitor of a cytokine receptor or cytokine selected fromamong IL-10, IL-10R, IL-6, IL-6 receptor, IFNγ, IFNGR, IL-2, IL-2R/CD25,MCP-1, CCR2, CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1, andIL-1Ralpha/IL-1beta; or an agent capable of preventing, blocking orreducing microglial cell activity or function.

In some embodiments, the agent capable of preventing, blocking orreducing microglial cell activity or function is selected from ananti-inflammatory agent, an inhibitor of NADPH oxidase (NOX2), a calciumchannel blocker, a sodium channel blocker, inhibits GM-CSF, inhibitsCSF1R, specifically binds CSF-1, specifically binds IL-34, inhibits theactivation of nuclear factor kappa B (NF-κB), activates a CB₂ receptorand/or is a CB₂ agonist, a phosphodiesterase inhibitor, inhibitsmicroRNA-155 (miR-155) or upregulates microRNA-124 (miR-124). In somecases, the agent is selected from minocycline, naloxone, nimodipine,Riluzole, MOR103, lenalidomide, a cannabinoid (optionally WIN55 or212-2), intravenous immunoglobulin (IVIg), ibudilast, anti-miR-155locked nucleic acid (LNA), MCS110, PLX-3397, PLX647, PLX108-D1, PLX7486,JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl) pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945, emactuzumab, IMC-CS4, FPA008,LY-3022855, AMG-820 and TG-3003. In some embodiments, the agent is aninhibitor of colony stimulating factor 1 receptor (CSF1R). For example,the agent PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527,JNJ28312141, ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945 or a pharmaceutical salt or prodrugthereof; emactuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003or is an antigen-binding fragment thereof, or a combination of any ofthe foregoing.

In some embodiments, the articles of manufacture and/or kits furtherinclude one or more reagents for assaying biological samples, e.g.,biological samples from subjects who are candidates for administrationor who have been administered the therapy, and optionally instructionsfor use of the reagents or assays. In some embodiments, the biologicalsample is or is obtained from a blood, plasma or serum sample In someembodiments, the reagents can be used prior to the administration of thecell therapy or after the administration of cell therapy, for diagnosticpurposes, to identify subjects and/or to assess treatment outcomesand/or toxicities. For example, in some embodiments, the article ofmanufacture and/or kits further contain reagents for measuring the levelof particular biomarkers, e.g., cytokines or analytes, that areassociated with toxicity, and instructions for measuring. In someembodiments, the reagents include components for performing an in vitroassay to measure the biomarkers (e.g. analytes), such as an immunoassay,an aptamer-based assay, a histological or cytological assay, or an mRNAexpression level assay. In some embodiments, the in vitro assay isselected from among an enzyme linked immunosorbent assay (ELISA),immunoblotting, immunoprecipitation, radioimmunoassay (RIA),immunostaining, flow cytometry assay, surface plasmon resonance (SPR),chemiluminescence assay, lateral flow immunoassay, inhibition assay andavidity assay. In some aspects, the reagent is a binding reagent thatspecifically binds the biomarkers (e.g. analytes). In some cases, thebinding reagent is an antibody or antigen-binding fragment thereof, anaptamer or a nucleic acid probe.

In some embodiments, the articles of manufacture and/or kits compriseone or more reagent capable of detecting one or more analytes, andinstructions for using the reagent to assay a biological sample from asubject that is a candidate for treatment, wherein the one or moreanalytes is selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10,IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF,IL-1Ralpha, IL-1Rbeta, IP-10, perforin, and D-dimer (fibrin degradationproduct). In some embodiments, instructions for assaying presence orabsence, level, amount, or concentration of an analyte in the subjectcompared to a threshold level of the analyte is also included.

In some embodiments, the instructions are included which specify, if thelevel, amount or concentration of the analyte in the sample is at orabove a threshold level for the analyte, administering to the subject anagent or other treatment capable of treating, preventing, delaying,reducing or attenuating the development or risk of development of atoxicity (i) prior to, (ii) within one, two, or three days of, (iii)concurrently with and/or (iv) at first fever following, the initiationof administration of the cell therapy to the subject. In some cases, theinstructions specify that if the level, amount or concentration of theanalyte in the sample is at or above a threshold level for the analyte,the cell therapy is administered to the subject at a reduced dose or ata dose that is not associated with risk of developing toxicity or severetoxicity, or is not associated with a risk of developing a toxicity orsevere toxicity in a majority of subjects, and/or a majority of subjectshaving a disease or condition that the subject has or is suspected ofhaving, following administration of the cell therapy. In some cases, theinstructions specify that if the level, amount or concentration of theanalyte in the sample is at or above a threshold level for the analyte,the cell therapy is administered in an in-patient setting and/or withadmission to the hospital for one or more days, optionally wherein thecell therapy is otherwise to be administered to subjects on anoutpatient basis or without admission to the hospital for one or moredays.

In some embodiments, the instructions for administering the cell therapyspecify, if the level, amount or concentration of the analyte in thesample, is below a threshold level, administering to the subject thecell therapy, optionally at a non-reduced dose, optionally on anoutpatient basis or without admission to the hospital for one or moredays. In some embodiments, the instructions for administering the celltherapy specify, if the level, amount or concentration of the analyte inthe sample, is below a threshold level, prior to or concurrently withadministering the cell therapy and/or prior to the development of a signor symptom of a toxicity other than fever, an agent or treatment capableof treating, preventing, delaying, or attenuating the development of thetoxicity is not administered to the subject. In some aspects, theinstructions for administering the cell therapy specify that if thelevel, amount or concentration of the analyte in the sample, is below athreshold level, the administration of the cell therapy is to be or maybe administered to the subject on an outpatient setting and/or withoutadmission of the subject to the hospital overnight or for one or moreconsecutive days and/or is without admission of the subject to thehospital for one or more days.

The articles of manufacture and/or kits may further include a celltherapy and/or further include instructions for use with, prior toand/or in connection with treatment with the cell therapy. In someembodiments, the instructions are included for administering the agentand the instructions specify if the level, amount or concentration ofthe analyte in the sample, is at or above a threshold leveladministering to the subject the agent. In some aspects, theinstructions further specify administering a cell therapy to thesubject, wherein administration of the agent is to be carried out (i)prior to, (ii) within one, two, or three days of, (iii) concurrentlywith and/or (iv) at first fever following, the initiation ofadministration of the cell therapy to the subject.

The articles of manufacture and/or kits may include a container and alabel or package insert on or associated with the container. Suitablecontainers include, for example, bottles, vials, syringes, IV solutionbags, etc. The containers may be formed from a variety of materials suchas glass or plastic. The container in some embodiments holds acomposition which is by itself or combined with another compositioneffective for treating, preventing and/or diagnosing the condition. Insome embodiments, the container has a sterile access port. Exemplarycontainers include an intravenous solution bags, vials, including thosewith stoppers pierceable by a needle for injection, or bottles or vialsfor orally administered agents. The label or package insert may indicatethat the composition is used for treating a disease or condition. Thearticle of manufacture may include (a) a first container with acomposition contained therein, wherein the composition includesengineered cells expressing a recombinant receptor; and (b) a secondcontainer with a composition contained therein, wherein the compositionincludes the second agent. In some embodiments, the article ofmanufacture may include (a) a first container with a first compositioncontained therein, wherein the composition includes a subtype ofengineered cells expressing a recombinant receptor; and (b) a secondcontainer with a composition contained therein, wherein the compositionincludes a different subtype of engineered cells expressing arecombinant receptor. The article of manufacture may further include apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further include another or the same container comprisinga pharmaceutically-acceptable buffer. It may further include othermaterials such as other buffers, diluents, filters, needles, and/orsyringes.

VII. Exemplary Embodiments

Among the provided embodiments are:

1. A method of treating a subject having or suspected of having adisease or condition, the method comprising administering to the subjecta dose of CD4⁺ and CD8⁺ T cells, each of the CD4⁺ and the CD8⁺ T cells,individually, comprising a receptor that specifically binds to a targetantigen expressed by the disease or condition or a cell or tissuethereof and/or that is associated with the disease or condition, whereinthe administration comprises administering a plurality of separatecompositions, the plurality of separate compositions comprising a firstcomposition comprising one of the CD4⁺ T cells and the CD8⁺ T cells andadministration of a second composition comprising the other of the CD4⁺T cells and the CD8⁺ T cells.

2. The method of embodiment 1, wherein the receptor comprised by theCD4⁺ T cells and/or the receptor comprised by the CD8⁺ T cells comprisesa recombinant receptor and/or wherein the CD4⁺ T cells and/or the CD8⁺ Tcells are genetically engineered to express the receptor.

3. The method of embodiment 1 or embodiment 2, wherein:

the administration of the first composition and the administration ofthe second composition are carried out on the same day, are carried outbetween about 0 and about 12 hours apart, between about 0 and about 6hours apart or between about 0 to 2 hours apart; or

the initiation of administration of the first composition and theinitiation of administration of the second composition are carried outbetween about 1 minute and about 1 hour apart or between about 5 minutesand about 30 minutes apart.

4. The method of any of embodiments 1-3, wherein the first compositionand second composition are administered no more than 2 hours, no morethan 1 hour, no more than 30 minutes, no more than 15 minutes, no morethan 10 minutes or no more than 5 minutes apart.

5. The method of any of embodiments 1-4, wherein the first compositioncomprises the CD4⁺ T cells.

6. The method of any of embodiments 1-5, wherein the first compositioncomprises the CD8⁺ T cells.

7. The method of any of embodiments 1-6, wherein the initiation of theadministration of the first composition is carried out prior to theinitiation of the administration of the second composition.

8. The method of any of embodiments 1-7, wherein:

the dose of cells comprises a defined ratio of CD4⁺ cells expressing arecombinant receptor to CD8⁺ cells expressing a recombinant receptorand/or of CD4⁺ cells to CD8⁺ cells, which ratio optionally is or isapproximately 1:1 or is between approximately 1:3 and approximately 3:1;and/or

the CD4⁺ T cells comprising the receptor in the one of the first andsecond compositions and the CD8⁺ T cells comprising the receptor in theother of the first and second compositions are present at a definedratio, which ratio optionally is or is approximately 1:1 or is betweenapproximately 1:3 and approximately 3:1; and/or

the CD4⁺ T cells comprising the receptor and the CD8⁺ T cells comprisingthe receptor administered in the first and second compositions arepresent at a defined ratio, which ratio optionally is or isapproximately 1:1 or is between approximately 1:3 and approximately 3:1.

9. The method of embodiment 8, wherein the defined ratio is or isapproximately 1:1.

10. The method of any of embodiments 1-9, wherein the dose of T cells isadministered to the subject as a single dose or is administered only onetime within a period of two weeks, one month, three months, six months,1 year or more.

11. The method of any of embodiments 1-9, wherein the dose of T cells isadministered as a double dose comprising a first dose of the T cells anda consecutive dose of the T cells, wherein one or both of the first doseand the second dose comprises administration of the plurality ofcomposition of T cells.

12. The method of embodiment 11, wherein the consecutive dose isadministered at a point in time that is at least or more than about 7days or 14 days after and less than about 28 days after initiation ofthe administration of the first dose of cells.

13. The method of any of embodiments 1-12, wherein:

the dose of cells comprises between at or about 1×10⁵ and at or about5×10⁸ total recombinant receptor-expressing T cells or total T cells,between at or about 1×10⁵ and at or about 1×10⁸ total recombinantreceptor-expressing T cells or total T cells, between at or about 5×10⁵and at or about 1×10⁷ total recombinant receptor-expressing T cells ortotal T cells, or between at or about 1×10⁶ and at or about 1×10⁷ totalrecombinant receptor-expressing T cells or total T cells, eachinclusive.

14. The method of any of embodiments 1-13, wherein the dose of T cellscomprises the administration of no more than 1×10⁸ total recombinantreceptor-expressing T cells or total T cells, no more than 1×10⁷ totalrecombinant receptor-expressing T cells or total T cells, no more than0.5×10⁷ total recombinant receptor-expressing T cells or total T cells,no more than 1×10⁶ total recombinant receptor-expressing T cells ortotal T cells, no more than 0.5×10⁶ total recombinantreceptor-expressing T cells or total T cells.

15. The method of any of embodiments 1-14, wherein the dose of CD4⁺ andCD8⁺ T cells comprises:

between at or about 5×10⁷ recombinant receptor-expressing T cells and ator about 1×10⁸ recombinant receptor-expressing T cells, each inclusive;

between at or about 1×10⁷ and at or about 2×10⁸ recombinantreceptor-expressing T cells;

between at or about 5×10⁷ and at or about 1.5×10⁸ recombinantreceptor-expressing T cells

at or about 5×10⁷ recombinant receptor-expressing T cells; or

at or about 1×10⁸ recombinant receptor-expressing T cells; or

the dose comprises at or about 1.5×10⁸ recombinant receptor-expressing Tcells;

between at or about 5×10⁶ and at or about 1×10⁸ recombinantreceptor-expressing CD8⁺ T cells;

between at or about 1×10⁷ and at or about 0.75×10⁸ recombinantreceptor-expressing CD8⁺ T cells;

at or about 2.5×10⁷ recombinant receptor-expressing CD8⁺ T cells;

at or about 5×10⁷ recombinant receptor-expressing CD8⁺ T cells; and/orat or about 0.75×10⁸ recombinant receptor-expressing CD8⁺ T cells.

16. The method of any of embodiments 1-15, wherein the recombinantreceptor specifically binds to an antigen associated with the disease orcondition or expressed in cells of the environment of a lesionassociated with the disease or condition.

17. The method of any of embodiments 1-16, wherein the disease orcondition is a cancer.

18. The method of any of embodiments 1-17, wherein the disease orcondition is a myeloma, leukemia or lymphoma.

19. The method of any of embodiments 1-18, wherein the antigen is ROR1,B cell maturation antigen (BCMA), carbonic anhydrase 9 (CAIX), tEGFR,Her2/neu (receptor tyrosine kinase erbB2), L1-CAM, CD19, CD20, CD22,mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor,CD23, CD24, CD30, CD33, CD38, CD44, EGFR, epithelial glycoprotein 2(EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3,erb-B4, erbB dimers, EGFR vIII, folate binding protein (FBP), FCRLS,FCRHS, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha,IL-13R-alpha2, kinase insert domain receptor (kdr), kappa light chain,Lewis Y, L1-cell adhesion molecule, (L1-CAM), Melanoma-associatedantigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentially expressed antigen ofmelanoma (PRAME), survivin, TAG72, B7-H6, IL-13 receptor alpha 2(IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE A1, HLA-A2NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9,NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands, CD44v6, dualantigen, a cancer-testes antigen, mesothelin, murine CMV, mucin 1(MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100, oncofetal antigen,ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2/neu, estrogenreceptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2,O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2,CCL-1, CD138, G Protein Coupled Receptor 5D (GPCRSD), or apathogen-specific antigen.

20. The method of any of embodiments 1-19, wherein the antigen is CD19.

21. The method of any of embodiments 1-20, wherein the disease orcondition is a B cell malignancy and/or is acute lymphoblastic leukemia(ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkinlymphoma (NHL), and Diffuse Large B-Cell Lymphoma (DLBCL).

22. The method of any of embodiments 1-21, wherein the disease orcondition is NHL and the NHL is selected from the group consisting ofaggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS (de novo andtransformed from indolent), primary mediastinal large B cell lymphoma(PMBCL), T cell/histocyte-rich large B cell lymphoma (TCHRBCL),Burkitt's lymphoma, mantle cell lymphoma (MCL), and/or follicularlymphoma (FL), optionally follicular lymphoma Grade 3B (FL3B).

23. The method of any of embodiments 1-22, wherein the recombinantreceptor is a chimeric antigen receptor (CAR), optionally wherein therecombinant receptor is a chimeric antigen receptor (CAR), optionallywherein the CAR comprises an extracellular antigen-recognition domainthat specifically binds to the antigen and an intracellular signalingdomain comprising an ITAM, wherein optionally, the intracellularsignaling domain comprises an intracellular domain of a CD3-zeta (CD3ζ)chain; and/or wherein the CAR further comprises a costimulatorysignaling region, which optionally comprises a signaling domain of CD28or 4-1BB.

24. An article of manufacture, comprising (1) a vial comprising acomposition comprising a plurality of CD4⁺ T cells expressing arecombinant receptor, and (2) instructions for administering, to asubject having a disease or condition:

a unit dose of cells of the composition, the unit dose comprising all ora portion of the plurality of CD4⁺ T cells, and a unit dose of acomposition comprising CD8⁺ T cells expressing a recombinant receptor,wherein the vial does not comprise the unit dose of the compositioncomprising CD8+ T cells expressing the recombinant receptor.

25. An article of manufacture, comprising (1) a vial comprising acomposition comprising a plurality of CD8⁺ T cells expressing arecombinant receptor, and (2) instructions for administering, to asubject having a disease or condition:

a unit dose of cells of the composition, the unit dose comprising all ora portion of the plurality of CD8⁺ T cells, and a unit dose of acomposition comprising CD4⁺ T cells expressing a recombinant receptor,wherein the vial does not comprise the unit dose of the compositioncomprising CD4+ T cells expressing the recombinant receptor.

26. The article of manufacture of embodiment 24 or embodiment 25,wherein:

the recombinant receptor expressed by the CD4⁺ cells and the recombinantreceptor expressed by the CD8⁺ T cells is the same;

the recombinant receptor expressed by the CD4⁺ cells and the recombinantreceptor expressed by the CD8⁺ T cells is different; or

the recombinant receptor expressed by the CD4⁺ cells and the recombinantreceptor expressed by the CD8⁺ T cells binds to the same antigen, whichis expressed by or associated with the disease or condition or cell ortissue thereof.

27. The article of manufacture of any of embodiments 24-26, wherein thevial comprises greater than or greater than about 10×10⁶ T cells orrecombinant receptor-expressing T cells, greater than or greater thanabout 15×10⁶ T cells or recombinant receptor-expressing T cells, greaterthan or greater than about 25×10⁶ T cells or recombinantreceptor-expressing T cell.

28. The article of manufacture of any of embodiments 24-27, wherein thevial comprises between about 10 million cells per mL and about 70million cells per mL, between about 10 million cells per mL and about 50million cells per mL, between about 10 million cells per mL and about 25million cells per mL, between about 10 million cells per mL and about 15million cells per mL, 15 million cells per mL and about 70 million cellsper mL, between about 15 million cells per mL and about 50 million cellsper mL, between about 15 million cells per mL and about 25 million cellsper mL, between about 25 million cells per mL and about 70 million cellsper mL, between about 25 million cells per mL and about 50 million cellsper mL, and between about 50 million cells per mL and about 70 millioncells per mL.

29. The article of manufacture of any of embodiments 24-28, wherein thecomposition further comprise(s) a cryoprotectant and/or the articlefurther includes instructions for thawing the composition prior toadministration to the subject.

30. The article of manufacture of any of embodiments 24-29, wherein:

the plurality of CD4⁺ cells expressing the recombinant receptor and theplurality of CD8⁺ cells expressing the recombinant receptor in thearticle, and/or the CD4⁺ cells and the CD8⁺ cells in the vial, arepresent at a defined ratio, which optionally is at or is approximately1:1 or is between approximately 1:3 and approximately 3:1; and/or

the article further comprises information or instructions specifying theadministration of the plurality of cells at a defined ratio of cells ofthe plurality of CD4⁺ cells expressing the recombinant receptor andcells of the plurality of CD8⁺ cells expressing the recombinantreceptor, and/or the CD4⁺ cells and the CD8⁺ cells, which ratio isoptionally is at or is approximately 1:1 or is between approximately 1:3and approximately 3:1; and/or

the article further comprises information or instructions specifying theadministration of formulations in the vials at a defined volumetric orweight-based ratio, which optionally is at or about 1:1 and/or betweenat or about 3:1 and at or about 1:3, by volume or weight, and/oroptionally corresponds to a ratio of CD4⁺ cells expressing therecombinant receptor to CD8⁺ cells expressing the recombinant receptor,and/or CD4⁺ cells to CD8⁺ cells, which ratio is optionally is at or isapproximately 1:1 or is between approximately 1:3 and approximately 3:1.

31. The article of manufacture of embodiment 30, wherein the definedratio is or is approximately 1:1.

32. The article of manufacture of any of embodiments 24-31, wherein

the plurality of vials or plurality of cells or unit dose of cellsspecified for administration, collectively, comprises a dose of cellscomprising from or from about 1×10⁵ to 5×10⁸ total recombinantreceptor-expressing T cells or total T cells, 1×10⁵ to 1×10⁸ totalrecombinant receptor-expressing T cells or total T cells, from or fromabout 5×10⁵ to 1×10⁷ total recombinant receptor-expressing T cells ortotal T cells, or from or from about 1×10⁶ to 1×10⁷ total recombinantreceptor-expressing T cells or total T cells, each inclusive; thearticle comprises one or more unit dose of the CD4⁺ and CD8⁺ cells or ofthe CD4⁺receptor⁺ cells and CD8⁺ receptor⁺ cells, wherein the unit dosecomprises between at or about 1×10⁷ and at or about 2×10⁸ recombinantreceptor-expressing T cells, between at or about 5×10⁷ and at or about1.5×10⁸ recombinant receptor-expressing T cells, at or about 5×10⁷recombinant receptor-expressing T cells, at or about 1×10⁸ recombinantreceptor-expressing T cells, or at or about 1.5×10⁸ recombinantreceptor-expressing T cells, optionally wherein the information in thearticle specifies administration of one or of a plurality of unit dosesand/or a volume corresponding to such one or plurality of unit doses;

the article comprises one or more unit doses of the CD8⁺ cells, whereinthe dose comprises between at or about 5×10⁶ and at or about 1×10⁸recombinant receptor-expressing CD8⁺ T cells, the dose comprises betweenat or about 1×10⁷ and at or about 0.75×10⁸ recombinantreceptor-expressing CD8⁺ T cells, the dose comprises at or about 2.5×10⁷recombinant receptor-expressing CD8⁺ T cells, or the dose comprises ator about 5×10⁷ recombinant receptor-expressing CD8⁺ T cells, or the dosecomprises at or about 0.75×10⁸ recombinant receptor-expressing CD8⁺ Tcells, optionally wherein the information in the article specifiesadministration of one or of a plurality of unit doses and/or a volumecorresponding to such one or plurality of unit doses.

33. The article of manufacture of any of embodiments 24-32, wherein thecells in the article, collectively, comprise a dose of cells comprisingno more than 1×10⁸ total recombinant receptor-expressing T cells ortotal T cells, no more than 1×10⁷ total recombinant receptor-expressingT cells or total T cells, no more than 0.5×10⁷ total recombinantreceptor-expressing T cells or total T cells, no more than 1×10⁶ totalrecombinant receptor-expressing T cells or total T cells, no more than0.5×10⁶ total recombinant receptor-expressing T cells or total T cells.

34. The article of manufacture of any of embodiments 24-33, wherein theplurality of containers, collectively, comprises a dose of cellscomprising:

between at or about 5×10⁷ recombinant receptor-expressing T cells and1×10⁸ recombinant receptor-expressing T cells, each inclusive;

between at or about 1×10⁷ and at or about 2×10⁸ recombinantreceptor-expressing T cells;

between at or about 5×10⁷ and at or about 1.5×10⁸ recombinantreceptor-expressing T cells;

at or about 5×10⁷ recombinant receptor-expressing T cells;

at or about 1×10⁸ recombinant receptor-expressing T cells;

at or about 1.5×10⁸ recombinant receptor-expressing T cells;

between at or about 5×10⁶ and at or about 1×10⁸ recombinantreceptor-expressing CD8⁺ T cells;

between at or about 1×10⁷ and at or about 0.75×10⁸ recombinantreceptor-expressing CD8⁺ T cells;

at or about 2.5×10⁷ recombinant receptor-expressing CD8⁺ T cells;

at or about 5×10⁷ recombinant receptor-expressing CD8⁺ T cells; or at orabout 0.75×10⁸ recombinant receptor-expressing CD8⁺ T cells.

35. The article of manufacture of any of embodiments 24-34, wherein theinstructions specify administering all or a portion of the CD4⁺ T cellsand the all or a portion of the CD8⁺ T cells 0 to 12 hours apart, 0 to 6hours apart or 0 to 2 hours apart.

36. The article of manufacture of any of embodiments 24-34, wherein theinstructions specify administering the CD4⁺ T cells and the CD8⁺ T cellsno more than 2 hours, no more than 1 hour, no more than 30 minutes, nomore than 15 minutes, no more than 10 minutes or no more than 5 minutesapart.

37. The article of manufacture of any of embodiments 24-36, wherein theinstructions specify administering the CD4⁺ T cells prior toadministering the CD8⁺ cells.

38. The article of manufacture of any of embodiments 24-36, wherein theinstructions specify administering the CD8⁺ T cells prior toadministering the CD4⁺ Cells.

39. The article of manufacture of any of embodiments 24-38, wherein therecombinant receptor specifically binds to an antigen associated withthe disease or condition or expressed in cells of the environment of alesion associated with the disease or condition.

40. The article of manufacture of any of embodiments 24-39, wherein thedisease or condition is a cancer.

41. The article of manufacture of any of embodiments 24-40, wherein thedisease or condition is a myeloma, leukemia or lymphoma.

42. The article of manufacture of any of embodiments 24-41, wherein theantigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9(CAIX), tEGFR, Her2/neu (receptor tyrosine kinase erbB2), L1-CAM, CD19,CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen,anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR,epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40),EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate bindingprotein (FBP), FCRLS, FCRHS, fetal acetylcholine receptor, GD2, GD3,HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor(kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM),Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentiallyexpressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AIMAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8,avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2Dligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin,murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100,oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA),Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), acyclin, cyclin A2, CCL-1, CD138, G Protein Coupled Receptor 5D (GPCRSD),or a pathogen-specific antigen.

43. The article of manufacture of any of embodiments 24-42, wherein theantigen is CD19.

44. The article of manufacture of any of embodiments 24-43, wherein thedisease or condition is a B cell malignancy and/or is acutelymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia(CLL), non-Hodgkin lymphoma (NHL), and Diffuse Large B-Cell Lymphoma(DLBCL).

45. The article of manufacture of any of embodiments 24-44, wherein thedisease or condition is NHL and the NHL is selected from the groupconsisting of aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS(de novo and transformed from indolent), primary mediastinal large Bcell lymphoma (PMBCL), T cell/histocyte-rich large B cell lymphoma(TCHRBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL), and/orfollicular lymphoma (FL), optionally follicular lymphoma Grade 3B(FL3B).

46. The article of manufacture of any of embodiments 24-45, wherein theT cells are primary T cells obtained from a subject.

47. The article of manufacture of any of embodiments 24-46, wherein theT cells are autologous to the subject.

48. The article of manufacture of any of embodiments 24-46, wherein theT cells are allogeneic to the subject.

49. A method of treating a subject having or suspected of having a Bcell malignancy, optionally a non-Hodgkin lymphoma (NHL), the methodcomprising administering to the subject a dose of T cells comprising Tcells expressing a chimeric antigen receptor (CAR) that specificallybinds to a target antigen expressed by the NHL, wherein:

the dose of T cells comprises between at or about 2.5×10⁷ CAR-expressingT cells and 2×10⁸ CAR-expressing T cells, inclusive, optionally betweenat or about 5×10⁷ CAR-expressing T cells and at or about 1×10⁸CAR-expressing T cells, inclusive; and

the malignancy, optionally the NHL, comprises diffuse large B celllymphoma (DLBCL), primary mediastinal large B cell lymphoma (PMBCL), NOS(de novo or transformed from indolent lymphoma), or follicular lymphomaGrade 3B and wherein the subject is or has been identified as having anEastern Cooperative Oncology Group Performance Status (ECOG) status of 0or 1, optionally wherein the method further comprises identifying thesubject as having diffuse large B cell lymphoma (DLBCL), primarymediastinal large B cell lymphoma (PMBCL), NOS (de novo or transformedfrom indolent lymphoma), or follicular lymphoma Grade 3B having an ECOGstatus of 0 or 1.

50. The method of embodiment 49, wherein the dose of T cells comprises adefined ratio of CD4⁺ cells expressing the CAR to CD8⁺ cells expressingthe CAR and/or of CD4⁺ cells to CD8⁺ cells, which ratio optionally isapproximately 1:1 or is between approximately 1:3 and approximately 3:1.

51. A method of treating a subject having non-Hodgkin lymphoma (NHL),the method comprising administering to the subject a dose of T cellscomprising T cells expressing a chimeric antigen receptor (CAR) thatspecifically binds to a target antigen expressed by the NHL, the dose ofT cells comprising a defined ratio of CD4⁺ cells expressing the CAR toCD8⁺ cells expressing the CAR and/or of CD4⁺ cells to CD8⁺ cells, whichratio is approximately or is 1:1, wherein the NHL comprises diffuselarge B cell lymphoma (DLBCL), primary mediastinal large B cell lymphoma(PMBCL), NOS (de novo or transformed from indolent lymphoma), orfollicular lymphoma Grade 3B.

52. The method of embodiment 51, wherein the subject is or has beenidentified as having an Eastern Cooperative Oncology Group PerformanceStatus (ECOG) status of 0, 1 or 2.

53. The method of embodiment 51 or embodiment 52, wherein the subject isor has been identified as having an ECOG status of 0 or 1.

54. The method of any of embodiments 49-53, wherein:

at least 35%, at least 40% or at least 50% of subjects treated accordingto the method achieve a complete response (CR), optionally wherein theCR is durable, or is durable in at least 60%, 70%, 80%, 90%, or 95% ofsubjects achieving the CR, for at or greater than 3 months or at orgreater than 6 months; and/or

at least 60%, 70%, 80%, 90%, or 95% of subjects achieving a CR by onemonth and/or by three months remain in response, remain in CR, and/orsurvive or survive without progression, for greater at or greater than 3months and/or at or greater than 6 months and/or at greater than ninemonths; and/or

at least 50%, at least 60% or at least 70% of the subjects treatedaccording to the method achieve objective response (OR) optionallywherein the OR is durable, or is durable in at least 60%, 70%, 80%, 90%,or 95% of subjects achieving the OR, for at or greater than 3 months orat or greater than 6 months; and/or

at least 60%, 70%, 80%, 90%, or 95% of subjects achieving the OR remainin response or surviving for greater at or greater than 3 months and/orat or greater than 6 months; and/or

at least 40%, at least 50%, at least 60%, at least 70% of the subjectswho, at or prior to the administration of the dose of cells had or wereidentified to have a double/triple hit lymphoma or relapse, optionallyrelapse within 12 months, following administration of an autologous stemcell transplant (ASCT), achieved an OR, optionally wherein the OR isdurable for at or greater than 3 months or at or greater than 6 months.

55. The method of any of embodiments 49-54, wherein:

the cells are autologous to the subject and no minimum absolutelymphocyte count (ALC) for apheresis is required and/or specified forproduction of the therapy; and/or

the cells are produced by a process which, for at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of subjects having thedisease or condition or of the selected population of subjects, iscapable of generating a cell product for administration and outcomesaccording to the method.

56. The method of any of embodiment 49-55, wherein:

greater than or greater than about 50%, about 60%, about 70%, or about80% of the subjects treated according to the method do not exhibit agrade 3 or greater cytokine release syndrome (CRS) and/or do not exhibita grade 3 or greater neurotoxicity and/or greater than 40% or 50% or 55%do not exhibit any neurotoxicity or CRS.

57. A method of treating a subject having non-Hodgkin lymphoma (NHL),the method comprising administering to the subject a dose of T cellscomprising T cells expressing a chimeric antigen receptor (CAR) thatspecifically binds to a target antigen expressed by the NHL, wherein:

the dose of T cells comprises between at or about 5×10⁷ recombinantreceptor-expressing T cells and 1×10⁸ recombinant receptor-expressing Tcells, inclusive, said dose comprising a defined ratio of CD4⁺ cellsexpressing the recombinant receptor to CD8⁺ cells expressing therecombinant receptor and/or of CD4⁺ cells to CD8⁺ cells, which ratio isapproximately or is 1:1; and

the method results in (1) a complete response (CR) in at least 35%, atleast 40% or at least 50% of subjects treated and/or objective response(OR) in at least 50%, at least 60% or at least 70% of subjects treatedand (2) results in no more than 50% of subjects exhibiting a cytokinerelease syndrome (CRS) higher than grade 2 and/or a neurotoxicity higherthan grade 2.

58. The method of embodiment 57, wherein at least 40%, at least 50%, atleast 60%, at least 70% of the subjects who, at or prior to theadministration of the dose of cells had or were identified to have adouble/triple hit lymphoma or relapse following administration of anautologous stem cell transplant (ASCT), achieved an OR, optionallywherein the OR is durable for at or greater than 3 months or at orgreater than 6 months.

59. The method of embodiment 57, wherein:

the CR or the OR is durable for greater than 3 months or greater than 6months; and/or

at least 20%, at least 25%, at least 35%, at least 40% or at least 50%of subjects treated according to the method achieve a CR that isdurable; and/or

at least 60%, 70%, 80%, 90%, or 95% of subjects treated with the methodand who achieve a CR, remain in CR or remain in response or remainsurviving for at or greater than 3 months or at or greater than 6 monthsor at or greater than 9 months; and/or

at least 60%, 70%, 80%, 90%, or 95% of subjects treated with the methodwho achieve a CR by one month and/or by three months remain in response,remain in CR, and/or survive or survive without progression, for greaterat or greater than 3 months and/or at or greater than 6 months and/or atgreater than nine months; and/or

at least 50%, at least 60% or at least 70% of the subjects treatedaccording to the method achieve objective response (OR) optionallywherein the OR is durable, or is durable in at least 60%, 70%, 80%, 90%,or 95% of subjects achieving the OR, for at or greater than 3 months orat or greater than 6 months; and/or

at least 60%, 70%, 80%, 90%, or 95% of subjects treated with the methodand achieving an OR remain in response or surviving for greater at orgreater than 3 months and/or at or greater than 6 months.

60. The method of any of embodiments 49-59, wherein:

at or prior to administration of the dose of cells, the subject is orhas been identified as having a lymphoma associated with or involvingcentral nervous system (CNS) involvement; and/or

at least 70%, at least 80%, at least 90% or at least 95% of subjectstreated according to the method who, at or prior to the administrationof the dose of cells exhibited or were identified to exhibit a lymphomawith CNS involvement, achieved a resolution of the CNS disease.

61. A method of treating a subject, the method comprising administering,to a subject that has a lymphoma, a dose of T cells comprising T cellsexpressing a chimeric antigen receptor (CAR) that specifically binds toa target antigen expressed by the lymphoma, wherein the lymphoma in thesubject is associated with or involves central nervous system (CNS)involvement.

62. The method of embodiment 60 or embodiment 61, wherein, at or priorto the time of administration of the dose of cells, the subjectcomprises a brain lesion, optionally a temporal lobe brain lesion.

63. The method of embodiment 62, wherein the lymphoma is a B cellmalignancy.

64. The method of embodiment 61 or embodiment 62, wherein the lymphomais non-Hodgkin lymphoma (NHL).

65. The method of any of embodiments 60-64, wherein:

at least 35%, at least 40% or at least 50% of subjects treated accordingto the method achieve a complete response (CR) or remission of CNSdisease, optionally wherein the CR or remission of the CNS disease isdurable, or is durable in at least 60%, 70%, 80%, 90%, or 95% ofsubjects achieving the CR, for at or greater than 3 months or at orgreater than 6 months; and/or

at least 60%, 70%, 80%, 90%, or 95% of subjects achieving a CR orremission of CNS disease by one month and/or by three months remain inresponse, remain in CR, and/or survive or survive without progression,for greater at or greater than 3 months and/or at or greater than 6months and/or at greater than nine months; and/or

at least 50%, at least 60% or at least 70% of the subjects treatedaccording to the method achieve objective response (OR) or remission ofCNS disease optionally wherein the OR or remission of the CNS disease isdurable, or is durable in at least 60%, 70%, 80%, 90%, or 95% ofsubjects achieving the OR, for at or greater than 3 months or at orgreater than 6 months; and/or

at least 60%, 70%, 80%, 90%, or 95% of subjects achieving the OR orremission of CNS disease remain in response or surviving for greater ator greater than 3 months and/or at or greater than 6 months; and/or thebrain lesion is reduced in size or volume, optionally by greater than orgreater than about 25%, 50%, 75% or more; and/or reduction or remissionor clearance of CNS disease is achieved, optionally is achieved in

at least 35%, at least 40% or at least 50% of subjects treated accordingto the method.

66. The method of any of embodiments 49-65, wherein:

greater than or greater than about 30%, 35%, 40%, or 50% of the subjectstreated according to the method do not exhibit any grade of cytokinerelease syndrome (CRS) or neurotoxicity; and/or

at least at or about 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%of subjects treated according to the method do not exhibit early onsetCRS or neurotoxicity and/or do not exhibit onset of CRS earlier than 3days following initiation of the administration and/or do not exhibitonset of neurotoxicity earlier than 5 days following initiation of theadministration and/or wherein the median onset of neurotoxicity amongsubjects treated according to the method is at or after the median peakof, or median time to resolution of, CRS in subjects treated accordingto the method and/or the median onset of neurotoxicity among subjectstreated according to the method is greater than at or about 8, 9, 10, or11 days.

67. The method of any of embodiments 49-66, wherein:

prior to initiation of administration of the dose of cells, the subjecthas not been administered an agent or treatment capable of treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity; and/or

the subject is not administered an agent or treatment for the treatmentor prevention or reduction or attenuation of a neurotoxicity and/or acytokine release syndrome or risk thereof, within a period of timefollowing administration of the dose, which period of time is optionallyat or about 1, 2, 3, 4, 5 days, or is optionally at or about 6, 7, 8, 9,10, 11 days, or is optionally 1, 2, 3 or 4 weeks; and/or

the subject is not administered an agent or treatment for the treatmentor prevention or reduction or attenuation of a neurotoxicity and/or acytokine release syndrome or risk thereof, following administration ofthe dose, prior to or unless the subject exhibits a sign or symptom ofthe toxicity and/or prior to or unless the subject exhibits a sign orsymptom of the toxicity other than a fever, optionally wherein the feveris not a sustained fever or the fever is or has been reduced or reducedby more than 1° C. after treatment with an antipyretic; and/or

the administration and any follow-up is carried out on an outpatientbasis and/or without admitting the subject to a hospital and/or withoutan overnight stay at a hospital and/or without requiring admission to oran overnight stay at a hospital, optionally unless or until the subjectexhibits a sustained fever or a fever that is or has not been reduced ornot reduced by more than 1° C. after treatment with an antipyretic.

68. The method of any of embodiments 49-67, wherein:

prior to initiation of administration of the dose of cells, the subjecthas not been administered an anti-IL-6 or anti-IL-6R antibody,optionally tocilizumab or siltuximab, and/or has not been administered asteroid, optionally dexamethasone; and/or

the subject is not administered an anti-IL-6 or anti-IL-6R antibody,optionally tocilizumab or siltuximab, and/or has not been administered asteroid, optionally dexamethasone, within a period of time followingadministration of the dose, which period of time is optionally at orabout 1, 2, 3, 4, 5 days, or is optionally at or about 6, 7, 8, 9, 10,11 days, or is optionally 1, 2, 3 or 4 weeks; and/or

the subject is not administered an anti-IL-6 or anti-IL-6R antibody,optionally tocilizumab or siltuximab, and/or has not been administered asteroid, optionally dexamethasone, following administration of the celldose, prior to, or unless, the subject exhibits a sign or symptom of atoxicity, optionally a neurotoxicity or CRS, and/or prior to, or unless,the subject exhibits a sign or symptom of a toxicity, optionally aneurotoxicity or CRS, other than a fever, optionally wherein the feveris not a sustained fever or the fever is or has been reduced or reducedby more than 1° C. after treatment with an antipyretic; and/or

the administration and any follow-up is carried out on an outpatientbasis and/or without admitting the subject to a hospital and/or withoutan overnight stay at a hospital and/or without requiring admission to oran overnight stay at a hospital, optionally unless or until the subjectexhibits a sustained fever or a fever that is or has not been reduced ornot reduced by more than 1° C. after treatment with an antipyretic.

69. The method of any of embodiments 49-67, wherein:

the administration is carried out on an outpatient basis and/or withoutrequiring admission to or an overnight stay at a hospital; and

if the subject exhibits a sustained fever or a fever that is or has notbeen reduced or not reduced by more than 1° C. after treatment with anantipyretic, the subject is admitted to the hospital or to an overnightstay at a hospital and/or is administered an agent or treatment for thetreatment or prevention or reduction or attenuation of a neurotoxicityand/or a cytokine release syndrome or risk thereof 70. The method of anyof embodiments 49-60 and 64-69, wherein the NHL is selected from thegroup consisting of aggressive NHL, diffuse large B cell lymphoma(DLBCL), NOS (de novo and transformed from indolent), primarymediastinal large B cell lymphoma (PMBCL), mantle cell lymphoma (MCL),and/or follicular lymphoma (FL), optionally follicular lymphoma Grade 3B(FL3B).

71. The method of any of embodiments 49-60 and 64-70, wherein the NHLcomprises diffuse large B cell lymphoma (DLBCL), primary mediastinallarge B cell lymphoma (PMBCL), NOS (de novo or transformed from indolentlymphoma), or follicular lymphoma Grade 3B.

72. The method of any of embodiments 49-60 and 64-71, wherein the NHLcomprises DLBCL.

73. The method of any of embodiments 49-60 and 70-72, wherein the DLBCLdoes not comprise DLBCL transformed from MZL and CLL (Richter's) and/orthe subject administered the dose of cells has a DLBCL characterized asde novo or transformed from indolent and/or does not comprise a DLBCLtransformed from MZL and CLL.

74. The method of any of embodiments 49-60 and 64-73, wherein the NHLdoes not comprise PMBCL and/or the subject administered the dose ofcells does not comprise PMBCL.

75. The method of any of embodiments 57-73, wherein the subject is orhas been identified as having an Eastern Cooperative Oncology GroupPerformance Status (ECOG) status of 0, 1 or 2.

76. The method of any of embodiments 49-75, wherein the subject is orhas been identified as having an ECOG status of 0 or 1.

77. The method of any of embodiments 49-76, wherein, at or immediatelyprior to the time of the administration of the dose of cells the subjecthas relapsed following remission after treatment with, or becomerefractory to, one or more prior therapies for the NHL, optionally one,two or three prior therapies other than another dose of cells expressingthe CAR.

78. The method of any of embodiments 49-77, wherein, at or prior to theadministration of the dose of cells:

the subject is or has been identified as having a double/triple hitlymphoma; and/or

the subject is or has been identified as having a chemorefractorylymphoma, optionally a chemorefractory DLBCL; and/or

the subject has not achieved complete remission (CR) in response to aprior therapy; and/or

the subject has relapsed within 1 year or less than 1 year afterreceiving an autologous stem cell transplant (ASCT).

79. The method of any of embodiments 49-78, comprising, prior toadministration of the dose of cells, identifying or selecting a subjectfor the administration of the dose of cells that has:

a double/triple hit lymphoma;

a chemorefractory lymphoma, optionally a chemorefractory DLBCL;

not achieved complete remission (CR) in response to a prior therapy fortreating the malignancy, optionally the NHL; and/or

has relapsed within 1 year or less than 1 year after receiving anautologous stem cell transplant (ASCT); and/or

has a lymphoma associated with or involving central nervous system (CNS)involvement.

80. The method of any of embodiments 49-79, further comprisingadministration of an additional therapeutic agent or therapy, optionallyother than a cell therapy, optionally other than CAR⁺ T cell therapy.

81. The method of embodiment 80, wherein the additional therapeuticagent or therapy is for treating the NHL or malignancy and/or increasesthe persistence, activity and/or efficacy of the dose of cells.

82. The method of embodiment 80 or embodiment 81, wherein the additionaltherapeutic agent or therapy is administered if the subject does notexhibit a response, optionally does not exhibit a CR or OR, to the celltherapy within 1 month, within 2 months or within 3 months afteradministration of the dose of cells.

83. The method of embodiment 80 or embodiment 81, wherein the additionaltherapeutic agent or therapy is administered to a subject:

that is or has been identified to have stable or progressive disease(SD/PD) following treatment with a prior therapy, optionally a priortherapy with a chemotherapeutic agent;

that is or has been identified with an Eastern Cooperative OncologyGroup Performance Status (ECOG) status of 2;

that is or has been identified as having a transformed follicularlymphoma (tFL); or

that is or has been identified has having a DLBCL transformed from MZLand CLL. 84. The method of any of embodiments 80-83, comprising, priorto administration of the dose of cells or the additional therapeuticagent or therapy, identifying or selecting a subject for theadministration of the dose of cells that has:

stable or progressive disease (SD/PD) following treatment with a priortherapy, optionally a prior therapy with a chemotherapeutic agent;

an Eastern Cooperative Oncology Group Performance Status (ECOG) statusof 2;

a transformed follicular lymphoma (tFL); or

a DLBCL transformed from MZL and CLL.

85. The method of any of embodiments 80-84, wherein the additionaltherapeutic agent or therapy is administered prior to, with or at thesame time and/or subsequent to initiation of administration of the doseof cells.

86. The method of any of embodiments 49-85, wherein:

the CAR comprises an scFv specific for the antigen, a transmembranedomain, a cytoplasmic signaling domain derived from a costimulatorymolecule, which optionally is or comprises a 4-1BB, and a cytoplasmicsignaling domain derived from a primary signaling ITAM-containingmolecule, which optionally is or comprises a CD3zeta signaling domainand optionally further comprises a spacer between the transmembranedomain and the scFv;

the CAR comprises, in order, an scFv specific for the antigen, atransmembrane domain, a cytoplasmic signaling domain derived from acostimulatory molecule, which optionally is or comprises a 4-1BBsignaling domain, and a cytoplasmic signaling domain derived from aprimary signaling ITAM-containing molecule, which optionally is aCD3zeta signaling domain; or

the CAR comprises, in order, an scFv specific for the antigen, a spacer,a transmembrane domain, a cytoplasmic signaling domain derived from acostimulatory molecule, which optionally is a 4-1BB signaling domain,and a cytoplasmic signaling domain derived from a primary signalingITAM-containing molecule, which optionally is or comprises a CD3zetasignaling domain; and wherein:

the spacer is optionally a polypeptide spacer that (a) comprises orconsists of all or a portion of an immunoglobulin hinge or a modifiedversion thereof or comprises about 15 amino acids or less, and does notcomprise a CD28 extracellular region or a CD8 extracellular region, (b)comprises or consists of all or a portion of an immunoglobulin hinge,optionally an IgG4 hinge, or a modified version thereof and/or comprisesabout 15 amino acids or less, and does not comprise a CD28 extracellularregion or a CD8 extracellular region, or (c) is at or about 12 aminoacids in length and/or comprises or consists of all or a portion of animmunoglobulin hinge, optionally an IgG4, or a modified version thereof;or (d) has or consists of the sequence of SEQ ID NO: 1, a sequenceencoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,SEQ ID NO: 33, SEQ ID NO: 34, or a variant of any of the foregoinghaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto, or (e) comprisesor consists of the formula X₁PPX₂P, where X₁ is glycine, cysteine orarginine and X₂ is cysteine or threonine; and/or

the costimulatory domain comprises SEQ ID NO: 12 or a variant thereofhaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto; and/or

the primary signaling domain comprises SEQ ID NO: 13 or 14 or 15 havingat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity thereto; and/or

the scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), aCDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence ofGNTLPYTFG (SEQ ID NO: 37) and/or a CDRH1 sequence of DYGVS (SEQ ID NO:38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and/or aCDRH3 sequence of YAMDYWG (SEQ ID NO: 40) or wherein the scFv comprisesa variable heavy chain region of FMC63 and a variable light chain regionof FMC63 and/or a CDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, aCDRL3 sequence of FMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence ofFMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as orcompetes for binding with any of the foregoing, and optionally whereinthe scFv comprises, in order, a V_(H), a linker, optionally comprisingSEQ ID NO: 24, and a V_(L), and/or the scFv comprises a flexible linkerand/or comprises the amino acid sequence set forth as SEQ ID NO: 24.

87. The method of any of embodiments 49-86, wherein the antigen is a Bcell antigen, which optionally is CD19.

88. The method of any of embodiments 1-23 and 49-87, wherein, prior tothe administration, the subject has been preconditioned with alymphodepleting therapy comprising the administration of fludarabineand/or cyclophosphamide.

89. The method of any of embodiments 1-23 and 49-88, further comprising,immediately prior to the administration, administering a lymphodepletingtherapy to the subject comprising the administration of fludarabineand/or cyclophosphamide.

90. The method of embodiment 88 or embodiment 89, wherein thelymphodepleting therapy comprises administration of cyclophosphamide atabout 200-400 mg/m², optionally at or about 300 mg/m², inclusive, and/orfludarabine at about 20-40 mg/m², optionally 30 mg/m², daily for 2-4days, optionally for 3 days, or wherein the lymphodepleting therapycomprises administration of cyclophosphamide at about 500 mg/m².

91. The method of embodiment 88 or embodiment 89, wherein:

the lymphodepleting therapy comprises administration of cyclophosphamideat or about 300 mg/m² and fludarabine at about 30 mg/m²daily for 3 days;and/or

the lymphodepleting therapy comprises administration of cyclophosphamideat or about 500 mg/m² and fludarabine at about 30 mg/m²daily for 3 days.

92. The method of any of embodiments 49-91, wherein the administrationof the cell dose and/or the lymphodepleting therapy is carried out viaoutpatient delivery, optionally unless or until the subject exhibits asustained fever or a fever that is or has not been reduced or notreduced by more than 1° C. after treatment with an antipyretic.

93. The method of embodiment 92, wherein if the subject exhibits asustained fever or a fever that is or has not been reduced or notreduced by more than 1° C. after treatment with an antipyretic, thesubject is admitted to the hospital or to an overnight stay at ahospital and/or is administered an agent or treatment for the treatmentor prevention or reduction or attenuation of a neurotoxicity and/or acytokine release syndrome or risk thereof.

94. The method of any of embodiments 49-92, wherein the dose of cells isadministered parenterally, optionally intravenously.

95. The method of any of embodiments 49-94, wherein:

at least 40% or at least 50% of subjects treated according to the methodachieve complete remission (CR), exhibit progression-free survival (PFS)and/or overall survival (OS) of greater than at or about 3 months, 6months or 12 months;

on average, subjects treated according to the method exhibit a medianPFS or OS of greater than at or about 6 months, 12 months, or 18 months;and/or

the subject exhibits PFS or OS following therapy for at least at orabout 6, 12, 18 or more months.

96. The method of any of embodiments 1-23 and 49-95, wherein, at orabout 14 or 28 days after initiation of administration of the dose ofcells, the number of CAR⁺ T cells, optionally CAR⁺ CD8⁺ T cells and/orCAR⁺ CD4⁺ T cells, detectable in the blood of the subject, or in amajority of subjects so treated by the method, is greater than 1 cellsper greater than 5 cells per μL or greater than per 10 cells per μL.

97. The method of any of embodiments 1-23 and 49-96, wherein the T cellsare primary T cells obtained from a subject.

98. The method of any of embodiments 1-23 and 49-97, wherein the T cellsare autologous to the subject.

99. The method of any of embodiments 1-23 and 49-97, wherein the T cellsare allogeneic to the subject.

100. The method of any of embodiments 49-99, wherein the T cellscomprise CD4⁺ T cells expressing the CAR and CD8⁺ T cells expressing theCAR and the administration comprises administering a plurality ofseparate compositions, said plurality of separate compositionscomprising a first composition comprising one of the CD4⁺ T cells andthe CD8⁺ T cells and the second composition comprising the other of theCD4⁺ T cells or the CD8⁺ T cells.

101. The method of embodiment 100, wherein:

the first composition and second composition are administered 0 to 12hours apart, 0 to 6 hours apart or 0 to 2 hours apart or wherein theadministration of the first composition and the administration of thesecond composition are carried out on the same day, are carried outbetween about 0 and about 12 hours apart, between about 0 and about 6hours apart or between about 0 and 2 hours apart; and/or

the initiation of administration of the first composition and theinitiation of administration of the second composition are carried outbetween about 1 minute and about 1 hour apart or between about 5 minutesand about 30 minutes apart.

102. The method of embodiment 100 or embodiment 101, wherein the firstcomposition and second composition are administered no more than 2hours, no more than 1 hour, no more than 30 minutes, no more than 15minutes, no more than 10 minutes or no more than 5 minutes apart.

103. The method of any of embodiments 100-102, wherein the firstcomposition comprises the CD4⁺ T cells.

104. The method of any of embodiments 100-102, wherein the firstcomposition comprises the CD8⁺ T cells.

105. The method of any of embodiments 100-104, wherein the firstcomposition is administered prior to the second composition.

106. The method of any of embodiments 49-105, wherein the dose of Tcells is administered to the subject as a single dose or is administeredonly one time within a period of two weeks, one month, three months, sixmonths, 1 year or more.

107. The method of any of embodiments 49-106, wherein the dose of Tcells is administered as a double dose comprising a first dose of the Tcells and a consecutive dose of the T cells, wherein one or both of thefirst dose and the second dose comprises administration of the pluralityof compositions of T cells.

108. The method of embodiment 107, wherein the consecutive dose isadministered at a point in time that is at least or more than about 7days or 14 days after and less than about 28 days after initiation ofthe administration of the first dose of cells.

109. A method of assessing likelihood of a response to a cell therapy,the method comprising:

(a) assessing the level, amount or concentration of one or more analytein a biological sample, wherein the one or more analyte is selected fromferritin, LDH, CXCL10, G-CSF, and IL-10, wherein:

the biological sample is from a subject that is a candidate fortreatment with the cell therapy, said cell therapy comprising a dose ofgenetically engineered cells expressing a recombinant receptor; and

the biological sample is obtained from the subject prior toadministering the cell therapy and/or said biological sample does notcomprise the recombinant receptor and/or said engineered cells; and

(b) comparing, individually, the level, amount or concentration of theanalyte in the sample to a threshold level, thereby determining alikelihood that a subject will achieve a response to the cell therapy.

110. The method of embodiment 109, further comprising administering thecell therapy to the subject if the subject is likely to achieve aresponse.

111. A method of selecting a subject for treatment, the methodcomprising:

(a) assessing the level, amount or concentration of one or more analytein a biological sample, wherein the one or more analyte is selected fromferritin, LDH, CXCL10, G-CSF, and IL-10, wherein:

the biological sample is from a subject that is a candidate fortreatment with the cell therapy, said cell therapy comprising a dose ofgenetically engineered cells expressing a recombinant receptor; and

the biological sample is obtained from the subject prior toadministering the cell therapy and/or said biological sample does notcomprise the recombinant receptor and/or said engineered cells; and

(b) selecting a subject who is likely to respond to treatment based onthe results of determining a likelihood that a subject will achieve aresponse to the cell therapy by comparing, individually, the level,amount or concentration of the analyte in the sample to a thresholdlevel.

112. The method of embodiment 111, further comprising administering thecell therapy to the subject selected for treatment.

113. A method of treatment, the method comprising:

(a) selecting a subject who is likely to respond to treatment with acell therapy based on the results of determining a likelihood that asubject will achieve a response to the cell therapy by comparing,individually, the level, amount or concentration of one or more analytein a biological sample, wherein the one or more analyte is selected fromferritin, LDH, CXCL10, G-CSF, and IL-10, to a threshold level, wherein:

the biological sample is from a subject that is a candidate fortreatment with the cell therapy, said cell therapy comprising a dose ofgenetically engineered cells expressing a recombinant receptor; and thebiological sample is obtained from the subject prior to administeringthe cell therapy and/or said biological sample does not comprise therecombinant receptor and/or said engineered cells; and

(b) administering the cell therapy to a subject selected for treatment.

114. The method of any of embodiments 109-113, wherein:

the subject is likely to achieve a response if the level, amount orconcentration of one or more of the analyte is below a threshold leveland the subject is not likely to achieve a response if the level, amountor concentration of one or more of the analyte is above a thresholdlevel.

115. The method of any of embodiments 109-114, wherein the thresholdlevel is within 25%, within 20%, within 15%, within 10% or within 5%and/or is within a standard deviation below the median or mean level,amount or concentration, or is or is about the median or mean level,amount or concentration, of the analyte in a biological sample obtainedfrom a group of subjects prior to receiving a cell therapy, wherein eachof the subjects of the group went on to achieve a response afteradministration of a recombinant-receptor-expressing therapeutic cellcomposition for treating the same disease or condition.

116. The method of any of embodiments 109-114, wherein the thresholdlevel is within 25%, within 20%, within 15%, within 10% or within 5%and/or is within a standard deviation above the median or mean level,amount or concentration of the analyte in a biological sample obtainedfrom a group of subjects prior to receiving a cell therapy, wherein eachof the subjects of the group went on to exhibit stable disease (SD)and/or progressive disease (PD) after administration of arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

117. The method of any of embodiments 109-116, wherein the responsecomprises objective response.

118. The method of embodiment 117, wherein the objective responsecomprises complete response (CR) or partial response (PR).

119. A method of assessing likelihood of a durable response to a celltherapy, the method comprising:

(a) assessing the level, amount or concentration of one or more analytein a biological sample, wherein the one or more analyte is selected fromLDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α,IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β, wherein:

the biological sample is from a subject that is a candidate fortreatment with the cell therapy, said cell therapy comprising a dose ofgenetically engineered cells expressing a recombinant receptor; and

the biological sample is obtained from the subject prior toadministering the cell therapy and/or said biological sample does notcomprise the recombinant receptor and/or said engineered cells; and

(b) comparing, individually, the level, amount or concentration of theanalyte in the sample to a threshold level, thereby determining alikelihood that a subject will achieve a durable response to the celltherapy.

120. The method of embodiment 119, further comprising administering thecell therapy to the subject if the subject is likely to achieve aresponse.

121. A method of selecting a subject for treatment, the methodcomprising:

(a) assessing the level, amount or concentration of one or more analytein a biological sample, wherein the one or more analyte is selected fromLDH, ferritin, CRP, D-dimer, SAA-1, IL-6, IL-10, IL-15, IL-16, TNF-α,IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1 and MIP-1β, wherein:

the biological sample is from a subject that is a candidate fortreatment with the cell therapy, said cell therapy comprising a dose ofgenetically engineered cells expressing a recombinant receptor; and

the biological sample is obtained from the subject prior toadministering the cell therapy and/or said biological sample does notcomprise the recombinant receptor and/or said engineered cells; and

(b) selecting a subject who is likely to respond to treatment based onthe results of determining a likelihood that a subject will achieve adurable response to the cell therapy by comparing, individually, thelevel, amount or concentration of the analyte in the sample to athreshold level.

122. The method of embodiment 121, further comprising administering thecell therapy to the subject selected for treatment.

123. A method of treatment, the method comprising:

(a) selecting a subject who is likely to respond to treatment with acell therapy based on the results of determining a likelihood that asubject will achieve a durable response to the cell therapy bycomparing, individually, the level, amount or concentration of one ormore analyte in a biological sample to a threshold level, wherein theone or more analyte is selected from LDH, ferritin, CRP, D-dimer, SAA-1,IL-6, IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α, CXCL-10, IL-8, MCP-1and MIP-1β, wherein:

the biological sample is from a subject that is a candidate fortreatment with the cell therapy, said cell therapy comprising a dose ofgenetically engineered cells expressing a recombinant receptor; and

the biological sample is obtained from the subject prior toadministering the cell therapy and/or said biological sample does notcomprise the recombinant receptor and/or said engineered cells; and

(b) administering the cell therapy to a subject selected for treatment.

124. The method of any of embodiments 120-123, wherein:

the subject is likely to achieve a durable response if the level, amountor concentration one or more of the analyte is below a threshold leveland the subject is not likely to achieve a durable response if thelevel, amount or concentration one or more of the analyte is above athreshold level.

125. The method of any of embodiments 120-124, wherein the thresholdlevel is within 25%, within 20%, within 15%, within 10% or within 5%and/or is within a standard deviation below the median or mean level,amount or concentration, or is or is about the median or mean level,amount or concentration, of the analyte in a biological sample obtainedfrom a group of subjects prior to receiving a cell therapy, wherein eachof the subjects of the group went on to achieve a durable response afteradministration of a recombinant-receptor-expressing therapeutic cellcomposition for treating the same disease or condition.

126. The method of any of embodiments 120-124, wherein the thresholdlevel is within 25%, within 20%, within 15%, within 11% or within 5%and/or is within a standard deviation above the median or mean level,amount or concentration of the analyte in a biological sample obtainedfrom a group of subjects prior to receiving a cell therapy, wherein eachof the subjects of the group did not achieve a durable response afteradministration of a recombinant-receptor-expressing therapeutic cellcomposition for treating the same disease or condition.

127. The method of any of embodiments 120-126, wherein the durableresponse comprises a complete response (CR) or partial response (PR)that is durable for at or greater than 3 months, 4 months, 5 months, or6 months.

128. The method of any of embodiments 120-127, wherein the durableresponse comprises a CR or PR that is durable for at least 3 months.

129. A method of assessing the risk of developing a toxicity afteradministration of a cell therapy, the method comprising:

(a) assessing the level, amount or concentration of one or more analytein a biological sample from a subject or a volumetric measure of tumorburden in a subject, wherein the one or more analyte is selected fromLDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrin degradationproduct), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP-1αand MIP-1β, wherein:

the subject is a candidate for treatment with the cell therapy, saidcell therapy optionally comprising a dose or composition of geneticallyengineered cells expressing a recombinant receptor; and

the biological sample is obtained from the subject prior toadministering the cell therapy and/or said biological sample does notcomprise the recombinant receptor and/or said engineered cells; and

(b) comparing, individually, the level, amount or concentration of theanalyte in the sample or the volumetric measure of tumor burden to athreshold level, thereby determining a risk of developing a toxicityafter administration of the cell therapy.

130. A method of identifying a subject, the method comprising:

(a) assessing the level, amount or concentration of one or more analytein a biological sample from a subject or a volumetric measure of tumorburden in a subject, wherein the one or more analyte is selected fromLDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrin degradationproduct), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP-1αand MIP-1β, wherein:

the subject is a candidate for treatment with the cell therapy, saidcell therapy optionally comprising a dose or composition of geneticallyengineered cells expressing a recombinant receptor; and

the biological sample is obtained from the subject prior toadministering the cell therapy and/or said biological sample does notcomprise the recombinant receptor and/or said engineered cells; and

(b) identifying a subject who has a risk of developing a toxicity afteradministration of a cell therapy based by comparing, individually, thelevel, amount or concentration of the analyte in the sample or thevolumetric measure of tumor burden to a threshold level.

131. A method of treatment, comprising:

(a) assessing the level, amount or concentration of one or more analytein a biological sample from a subject or a volumetric measure of tumorburden in the subject, wherein the one or more analyte is selected fromLDH, Ferritin, C-reactive protein (CRP), D-dimer (fibrin degradationproduct), IL-6, IL-8, IL-10, IL-15, IL-16 TNF-α, IFN-α2, MCP-1, MIP-1αand MIP-1β, wherein:

the subject is a candidate for treatment with the cell therapy, saidcell therapy optionally comprising a dose or composition of geneticallyengineered cells expressing a recombinant receptor; and

the biological sample is obtained from the subject prior toadministering the cell therapy and/or said biological sample does notcomprise the recombinant receptor and/or said engineered cells; and; and

(b) comparing, individually, the level, amount or concentration of theanalyte in the sample or the volumetric measure of tumor burden to athreshold level, thereby determining a risk of developing a toxicityafter administration of the cell therapy; and

(c) following or based on the results of the assessment, administeringto the subject the cell therapy, and, optionally, an agent or othertreatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity.

132. The method of any of embodiments 129-131, wherein the biologicalsample is a blood or plasma sample.

133. The method of any of embodiments 129-132, wherein the volumetricmeasure of tumor burden is a sum of product dimensions (SPD) or is avolumetric measurement based on CT and/or MRI imaging or other imagingof body.

134. The method of embodiment 133, wherein the volumetric measure oftumor burden is carried out prior to treatment, prior to apheresis, orprior to cell product manufacturing.

135. The method of any of embodiments 129-134, further comprisingmonitoring the subject for symptoms of toxicity if the subject isadministered a cell therapy and is identified as having a risk ofdeveloping a toxicity.

136. The method of any of embodiments 129-135, wherein:

the subject has a risk of developing a toxicity if the level, amount orconcentration one or more of the analyte or the volumetric measure oftumor burden is above a threshold level and the subject has a low riskof developing a toxicity if the level, amount or concentration one ormore of the analyte or the volumetric measure of tumor burden is below athreshold level.

137. The method of any of embodiments 129-136, wherein the thresholdlevel is within 25%, within 20%, within 15%, within 10% or within 5%and/or is within a standard deviation above the median or mean level,amount or concentration, or is or is about the median or mean level,amount or concentration, of the analyte or the volumetric measure oftumor burden in a biological sample obtained from a group of subjectsprior to receiving a cell therapy, wherein each of the subjects of thegroup went on not to develop any toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

138. The method of any of embodiments 129-137, wherein the thresholdlevel is within 25%, within 20%, within 15%, within 10% or within 5%and/or is within a standard deviation below the median or mean level,amount or concentration of the analyte or the volumetric measure oftumor burden in a biological sample obtained from a group of subjectsprior to receiving a cell therapy, wherein each of the subjects of thegroup went on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

139. The method of any of embodiments 129-138, wherein the toxicity isneurotoxicity or CRS.

140. The method of any of embodiments 129-139, wherein the toxicity isgrade 1 or higher neurotoxicity or CRS.

141. The method of any of embodiments 129-140, wherein:

the toxicity is severe neurotoxicity or is grade 2 or higherneurotoxicit, a grade 3 or higher neurotoxicity, at least prolongedgrade 3 neurotoxicity or is at or above grade 4 or grade 5neurotoxixity; or

the toxicity is severe CRS or comprises grade 2 or higher or grade 3 orhigher CRS. 142. The method of any of embodiments 129-141, wherein thetoxicity is neurotoxicity and the volumetric measure of tumor burden isSPD and the one or more analyte is selected from LDH, IL-10, IL-15,IL-16, TNF-α and MIP-1β.

143. The method of any of embodiments 129-141, wherein the toxicity isneurotoxicity and one or more analytes is assessed and the analytes areselected from LDH, Ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2,MCP-1 and MIP-1β.

144. The method of any of embodiments 129-141, wherein the toxicity isneurotoxicity and one or more analytes is assessed and the analytes areselected from IL-8, IL-10 and CXCL10.

145. The method of embodiment 144, wherein the neurotoxicity is severeneurotoxicity or grade 3 or higher neurotoxicity.

146. The method of any of embodiments 129-141, wherein toxicity is CRSand the one or more analyte or volumetric measure of tumor burden isselected from LDH, SPD, CRP, d-dimer, IL-6, IL-15, TNF-α and MIP-1α.

147. The method of embodiment 146, wherein the CRS is severe CRS orgrade 3 or higher CRS.

148. The method of any of embodiments 129-147, wherein if the subject isidentified as having a risk of developing a toxicity, administering tothe subject:

(a) (1) an agent or other treatment capable of treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity and (2) the cell therapy, wherein administration of theagent is to be administered (i) prior to, (ii) within one, two, or threedays of, (iii) concurrently with and/or (iv) at first fever following,the initiation of administration of the cell therapy to the subject;and/or

(b) a cell therapy at a reduced dose or at a dose that is not associatedwith risk of developing toxicity or severe toxicity, or is notassociated with a risk of developing a toxicity or severe toxicity in amajority of subjects, and/or a majority of subjects having a disease orcondition that the subject has or is suspected of having, followingadministration of the cell therapy; and/or

(c) administering to the subject a cell therapy in an in-patient settingand/or with admission to the hospital for one or more days, optionallywherein the cell therapy is otherwise to be administered to subjects onan outpatient basis or without admission to the hospital for one or moredays.

149. The method of embodiment 148, wherein the agent or other treatmentis an anti-IL-6 antibody or an anti-IL6 receptor antibody.

150. The method of embodiment 149, wherein the agent or other treatmentis or comprises an agent selected from among tocilizumab, siltuximab,clazakizumab, sarilumab, olokizumab (CDP6038), elsilimomab,ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634, ARGX-109, FE301 andFM101.

151. The method of embodiment 148, wherein the agent or other treatmentis or comprises a steroid, optionally dexamethasone.

152. The method of any of embodiments 129-142 and 148-151, wherein avolumetric measure is assessed and the volumetric measure is SPD and thethreshold level is or is about 30 cm², is or is about 40 cm², is or isabout 50 cm², is or is about 60 cm², or is or is about 70 cm².

153. The method of embodiment 152, wherein the volumetric measure is SPDand the threshold level is or is about 50 cm².

154. The method of any of embodiments 129-151, wherein the one or moreanalyte is or comprises LDH and the threshold level is or is about 300units per liter, is or is about 400 units per liter, is or is about 500units per liter or is or is about 600 units per liter.

155. The method of any of embodiments 154, wherein the analyte is LDHand the threshold level is or is about 500 units per liter.

156. The method of any of embodiments 109-155, wherein the recombinantreceptor specifically binds to an antigen associated with the disease orcondition or expressed in cells of the environment of a lesionassociated with the disease or condition.

157. The method of any of embodiments 109-156, wherein the disease orcondition is a cancer.

158. The method of any of embodiments 109-157, wherein the disease orcondition is a myeloma, leukemia or lymphoma.

159. The method of any of embodiments 109-158, wherein the disease orcondition is a B cell malignancy and/or is acute lymphoblastic leukemia(ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkinlymphoma (NHL), and Diffuse Large B-Cell Lymphoma (DLBCL).

160. The method of any of embodiments 109-159, wherein the recombinantreceptor is a chimeric antigen receptor (CAR).

161. The method of any of embodiments 109-160, wherein the engineeredcells comprise T cells, optionally CD4⁺ and/or CD8+.

162. The method of embodiment 161, wherein the T cells are primary Tcells obtained from a subject or are autologous to the subject.

163. A method of selecting a subject for treatment, the methodcomprising:

(a) contacting a biological sample with one or more reagent capable ofdetecting or that is specific for one or more analyte, wherein the oneor more analyte is selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8,IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin,G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, and D-dimer (fibrindegradation product),

wherein:

the biological sample is from a subject that is a candidate fortreatment with a cell therapy, said cell therapy optionally comprising adose or composition of genetically engineered cells expressing arecombinant receptor; and

the biological sample is obtained from the subject prior toadministering the cell therapy and/or said biological sample does notcomprise the recombinant receptor and/or said engineered cells; and

(b) selecting a subject in which either:

(i) the level, amount or concentration of the analyte in the sample isat or above a threshold level, thereby identifying a subject that is atrisk for developing a toxicity to the cell therapy; or

(ii) the level, amount or concentration of the analyte is below athreshold level.

164. The method of embodiment 163, wherein:

(a) a subject in (i) is selected for administering to the subject (1) anagent or other treatment capable of treating, preventing, delaying,reducing or attenuating the development or risk of development of atoxicity and (2) the cell therapy, wherein administration of the agentis to be administered (i) prior to, (ii) within one, two, or three daysof, (iii) concurrently with and/or (iv) at first fever following, theinitiation of administration of the cell therapy to the subject; and/or

(b) a subject in (i) is selected for administering to the subject a celltherapy at a reduced dose or at a dose that is not associated with riskof developing toxicity or severe toxicity, or is not associated with arisk of developing a toxicity or severe toxicity in a majority ofsubjects, and/or a majority of subjects having a disease or conditionthat the subject has or is suspected of having, following administrationof the cell therapy; and/or

(c) a subject in (i) is selected for administering to the subject a celltherapy in an in-patient setting and/or with admission to the hospitalfor one or more days, optionally wherein the cell therapy is otherwiseto be administered to subjects on an outpatient basis or withoutadmission to the hospital for one or more days.

165. The method of embodiment 163 or embodiment 164, wherein a subjectin (i) is selected, and the method further comprises:

(a) administering to the subject (1) an agent or other treatment capableof treating, preventing, delaying, reducing or attenuating thedevelopment or risk of development of a toxicity and (2) the celltherapy, wherein administration of the agent is carried out (i) priorto, (ii) within one, two, or three days of, (iii) concurrently withand/or (iv) at first fever following, the initiation of administrationof the cell therapy to the subject; and/or

(b) administering to the subject a cell therapy at a reduced dose or ata dose that is not associated with risk of developing toxicity or severetoxicity, or is not associated with a risk of developing a toxicity orsevere toxicity in a majority of subjects, and/or a majority of subjectshaving a disease or condition that the subject has or is suspected ofhaving, following administration of the cell therapy; and/or

(c) administering to the subject a cell therapy or a dose of geneticallyengineered cells of a cell therapy that is not associated with risk ofdeveloping toxicity or severe toxicity, or is not associated with a riskof developing a toxicity or severe toxicity in a majority of subjects,and/or a majority of subjects having a disease or condition that thesubject has or is suspected of having, following administration of thecell therapy; and/or

(d) administering to the subject a cell therapy in an in-patient settingand/or with admission to the hospital for one or more days, optionallywherein the cell therapy is otherwise to be administered to subjects onan outpatient basis or without admission to the hospital for one or moredays.

166. The method of embodiment 163 or embodiment 164, wherein:

(a) a subject in (ii) is selected for administering to the subject acell therapy, optionally at a non-reduced dose, optionally on anoutpatient basis or without admission to the hospital for one or moredays;

(b) a subject in (ii) is selected for administering to the subject acell therapy, wherein the cell therapy does not comprise administering,prior to or concurrently with administering the cell therapy and/orprior to the development of a sign or symptom of a toxicity other thanfever, an agent or treatment capable of treating, preventing, delaying,or attenuating the development of the toxicity; and/or

(c) a subject in (ii) is selected for administering a cell therapy on anoutpatient setting and/or without admission of the subject to thehospital overnight or for one or more consecutive days and/or is withoutadmission of the subject to the hospital for one or more days.

167. The method of embodiment 163, 164 or 166, wherein a subject in (ii)is selected, and the method further comprises administering to thesubject the cell therapy, optionally at a non-reduced dose, optionallyon an outpatient basis or without admission to the hospital for one ormore days.

168. The method of any of embodiments 163, 164, 166 and 167, wherein asubject in (ii) is selected, and the method further comprisesadministering to the subject the cell therapy, wherein:

the administration of the cell therapy does not comprise administering,prior to or concurrently with administering the cell therapy and/orprior to the development of a sign or symptom of a toxicity other thanfever, an agent or treatment capable of treating, preventing, delaying,or attenuating the development of the toxicity; and/or

the administration of the cell therapy is to be or may be administeredto the subject on an outpatient setting and/or without admission of thesubject to the hospital overnight or for one or more consecutive daysand/or is without admission of the subject to the hospital for one ormore days.

169. A method of treatment, comprising:

(a) assaying a biological sample for the level, amount or concentrationof one or more analyte, wherein the biological sample is from a subjectthat is a candidate for treatment, optionally with a cell therapy, saidcell therapy optionally comprising a dose or composition of geneticallyengineered cells expressing a recombinant receptor for treating adisease or condition, wherein the one or more analyte is selected fromLDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, TNF-alpha,IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta,IP-10, perform, and D-dimer (fibrin degradation product); and

(b) following or based on the results of the assay, administering to thesubject the cell therapy, and, optionally, an agent or other treatmentcapable of treating, preventing, delaying, reducing or attenuating thedevelopment or risk of development of a toxicity.

170. A method of treatment of a subject having or suspected of having adisease or condition, the method comprising:

(1) administering to the subject a cell therapy comprising a dose orcomposition of genetically engineered expressing a recombinant receptorfor treating or that specifically recognizes the disease or condition,and, optionally, (2) further administering to the subject an agent orother treatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity,wherein:

the administration is carried out following, or is carried out in amanner based on the results of, assessment of one or more pre-treatmentparameters, wherein:

(a) the one or more pre-treatment parameters comprise a parameterassociated or correlated with, or that is a surrogate for, burden of thedisease or condition in the subject, which parameters optionallycomprise one or more analytes and/or one or more volumetric or otherbody measurement,

(b) the assessment of the one or more pre-treatment parameters comprisesassessment of an amount or level of, in a biological sample that is oris from a sample obtained from the subject, a level, amount orconcentration of one or more analyte, the biological sample is obtainedfrom the subject prior to administering the cell therapy, wherein thesample is obtained from the subject prior to the administration of thecell therapy and, optionally, prior to apheresis or prior to manufactureof cells for administration, and/or the one or more analyte isassociated with disease burden and/or the one or more analyte isselected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16,TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha,IL-1Rbeta, IP-10, perforin, and D-dimer (fibrin degradation product);and/or

(c) the assessment of the one or more pre-treatment parametersoptionally comprises assessment of a volumetric measurement of thedisease or condition in the subject, which optionally is a measurementobtained by imaging, optionally a sum of product dimensions (SPD)result, or other volumetric measurement(s), optionally based on CTand/or MRI imaging or other imaging of the body, carried out prior totreatment and optionally prior to apheresis or manufacturing of cells.

171. The method of embodiment 170, wherein said assessment of one ormore pre-treatment parameters comprises the assaying of one or moreanalytes, which comprises detection which optionally comprisescontacting a reagent capable of directly or indirectly detecting theanalyte(s) with the biological sample and determining the level, amountor concentration of the analyte(s) in the biological sample.

172. The method of embodiment 170 or embodiment 171, wherein theassessment of one or more pre-treatment parameters comprises (A)assessment of, in a biological sample that is or is from a sampleobtained from the subject prior to treatment and/or prior to apheresisor cell processing, a level, amount or concentration of one or moreanalytes, optionally associated with disease burden, and (B) assessmentof a volumetric measure of disease burden, wherein the measure ofdisease burden optionally is a sum of product dimensions (SPD) result,or other volumetric measurement(s), optionally based on CT and/or MRIimaging or other imaging of the body and/or is carried out prior totreatment, prior to apheresis, or prior to cell product manufacturing.

173. The method of any of embodiments 169-172, wherein, if the level,amount or concentration and/or measure, or combination thereof,resulting from or obtained via the pre-treatment assessment, is at orabove a threshold level:

the administering comprises the further administering to the subject theagent or other treatment capable of treating, preventing, delaying,reducing or attenuating the development or risk of development of atoxicity, optionally wherein said further administering is carried out(i) prior to, (ii) within one, two, or three days of, or within fourfive or six days of, (iii) concurrently with and/or (iv) at first feverfollowing, the initiation of administration of the cell therapy to thesubject; and/or

the cell therapy administered to the subject is administered (A) at areduced dose as compared to if the level, amount or concentration and/ormeasure, or combination thereof is below the threshold level, or (B) ata dose that is not associated with risk of developing toxicity or severetoxicity, or is not associated with a risk of developing a toxicity orsevere toxicity in a majority of subjects, and/or a majority of subjectshaving a disease or condition that the subject has or is suspected ofhaving, following administration of the cell therapy, or (C) at a dosereduced compared to the maximum tolerated dose or the approved dose ormaximum approved dose, for use in subjects having the disease orcondition or for treatment of the disease or condition; and/or

the administering to the subject of the cell therapy is carried out oris specified to be carried out in an in-patient setting and/or withadmission to the hospital for one or more days, optionally wherein thecell therapy is otherwise, if the level, amount, concentration, measureor combination is below the threshold or is not assessed, to beadministered to subjects on an outpatient basis or without admission tothe hospital for one or more days or overnight.

174. The method of any of embodiments 169-173, wherein, if the level,amount or concentration and/or measure, or combination thereof,resulting from or obtained via the pre-treatment assessment, is below athreshold level:

the administration of the cell therapy does not comprise administering,prior to or concurrently with administering the cell therapy and/orprior to the development of a sign or symptom of a toxicity other thanfever, an agent or treatment capable of treating, preventing, delaying,or attenuating the development of the toxicity; and/or

the administration of the cell therapy is to be, or may be, administeredto the subject on an outpatient setting and/or without admission of thesubject to the hospital overnight or for one or more consecutive daysand/or is without admission of the subject to the hospital for one ormore days; and/or

the cell therapy administered to the subject is administered (A) at adose, optionally an amount of T cells or engineered T cells or T cellsof a specified phenotype, that is higher than that that would beadministered if the level, amount or concentration and/or measure, orcombination thereof were at or above the threshold level, or (B) at adose, optionally an amount of T cells or engineered T cells or T cellsof a specified phenotype, that is or is about the maximum tolerated doseor the maximum dose, approved for use to treat subjects having thedisease or condition.

175. A method of prophylactic treatment, comprising administering, to asubject, an agent or other treatment capable of treating, preventing,delaying, reducing or attenuating the development or risk of developmentof a toxicity, wherein:

the subject is a candidate for treatment optionally with a cell therapy,said cell therapy optionally comprising a dose or composition ofgenetically engineered cells expressing a recombinant receptor fortreating a disease or condition; and

the subject has been identified as at risk for developing a toxicityfollowing or based on an assessment of one or more pre-treatmentparameters, wherein:

(a) the one or more pre-treatment parameters comprise a parameterassociated or correlated with, or that is a surrogate for, burden of thedisease or condition in the subject, which parameters optionallycomprise one or more analytes and/or one or more volumetric or otherbody measurement, or

(b) the assessment of the one or more pre-treatment parameters comprisesassessment of an amount or level of, in a biological sample that is oris from a sample obtained from the subject, a level, amount orconcentration of one or more analytes in a biological sample obtainedfrom or from a sample from a subject, for the level, amount orconcentration of one or more analyte, said biological sample obtainedfrom the subject prior to administering the cell therapy and/or saidbiological sample not comprising the recombinant receptor and/or saidengineered cells, wherein:

(i) the one or more pre-treatment parameters comprise a parameterassociated or correlated with, or that is a surrogate for, burden of thedisease or condition in the subject, which parameters optionallycomprise one or more analytes and/or one or more volumetric or otherbody measurement,

(ii) the assessment of the one or more pre-treatment parameterscomprises assessment of an amount or level of, in a biological samplethat is or is from a sample obtained from the subject, a level, amountor concentration of one or more analyte, the biological sample isobtained from the subject prior to administering the cell therapy,wherein the sample is obtained from the subject prior to theadministration of the cell therapy and, optionally, prior to apheresisor prior to manufacture of cells for administration, and/or the one ormore analyte is associated with disease burden and/or the one or moreanalyte is selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10,IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF,IL-1Ralpha, IL-1Rbeta, IP-10, perforin, and D-dimer (fibrin degradationproduct); and/or

(ii) the assessment of the one or more pre-treatment parametersoptionally comprises assessment of a volumetric measurement of thedisease or condition in the subject, which optionally is a measurementobtained by imaging, optionally a sum of product dimensions (SPD)result, or other volumetric measurement(s), optionally based on CTand/or MM imaging or other imaging of the body, carried out prior totreatment and optionally prior to apheresis or manufacturing of cells;or

wherein the one or more analyte is selected from LDH, ferritin, CRP,IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1,MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, andD-dimer (fibrin degradation product).

176. The method of embodiment 175, wherein said assay comprisesdetection which optionally comprises contacting a reagent capable ofdirectly or indirectly detecting the analyte with the biological sampleand determining the level, amount or concentration of the analyte in thebiological sample, and/or wherein the agent is administered to thesubject if the level, amount or concentration of the analyte in thesample is at or above a threshold level and optionally wherein the agentis administered (i) prior to, (ii) within one, two, or three days of,(iii) concurrently with and/or (iv) at first fever following, theinitiation of administration of the cell therapy to the subject.

177. The method of embodiment 175 or embodiment 176, wherein theassessment of one or more pre-treatment parameters comprises (A)assessment of, in a biological sample that is or is from a sampleobtained from the subject prior to treatment and/or prior to apheresisor cell processing, a level, amount or concentration of one or moreanalytes, optionally associated with disease burden, and (B) assessmentof a volumetric measure of disease burden, wherein the measure ofdisease burden optionally is a sum of product dimensions (SPD) result,or other volumetric measurement(s), optionally based on CT and/or Millimaging or other imaging of the body and/or is carried out prior totreatment, prior to apheresis, or prior to cell product manufacturing.

178. The method of any of embodiments 169-177, wherein the thresholdlevel is within 25%, within 20%, within 15%, within 10% or within 5% ofthe average level, amount or concentration or measure, and/or is withina standard deviation of the average level, amount or concentration ormeasure, of the analyte or parameter in a biological sample obtainedfrom a group of subjects prior to receiving a recombinantreceptor-expressing therapeutic cell composition, wherein each of thesubjects of the group went on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

179. The method of any of embodiments 169-178, wherein the reagent is abinding molecule that specifically binds to the analyte.

180. The method of any of embodiments 169-179, wherein the reagent is anantibody or an antigen-binding fragment thereof.

181. The method of any of embodiments 169-180, wherein the biologicalsample is or is obtained from a blood, plasma or serum sample.

182. The method of any of embodiments 169-181, wherein assaying orassessing cells the analyte comprises an immunoassay.

183. The method of any of embodiments 169-182, wherein the toxicitycomprises neurotoxicity or cytokine release syndrome (CRS), optionallygrade 1 or higher neurotoxicity or CRS.

184. The method of any of embodiments 169-183, wherein:

the toxicity comprises severe neurotoxicity and/or comprises a grade 2or higher neurotoxicity, a grade 3 or higher neurotoxicity, at leastprolonged grade 3 neurotoxicity or is at or above grade 4 or grade 5neurotoxicity; and/or

the toxicity comprises severe CRS and/or comprises grade 2 or higher orgrade 3 or higher CRS.

185. The method of any of embodiments 169-184, wherein the toxicity isassociated with cerebral edema.

186. The method of any of embodiments 169-185, wherein the agent orother treatment is or comprises one or more of a steroid; an antagonistor inhibitor of a cytokine receptor or cytokine selected from amongIL-10, IL-10R, IL-6, IL-6 receptor, IFNγ, IFNGR, IL-2, IL-2R/CD25,MCP-1, CCR2, CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1, andIL-1Ralpha/IL-1beta; or an agent capable of preventing, blocking orreducing microglial cell activity or function.

187. The method of embodiment 186, wherein the antagonist or inhibitoris or comprises an agent selected from among an antibody orantigen-binding fragment, a small molecule, a protein or peptide and anucleic acid.

188. The method of any of embodiments 169-187, wherein the agent orother treatment is an anti-IL-6 antibody or an anti-IL6 receptorantibody.

189. The method of any of embodiments 169-188, wherein the agent orother treatment is or comprises an agent selected from amongtocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab (CDP6038),elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136), CPSI-2634,ARGX-109, FE301 and FM101.

190. The method of any of embodiments 169-189, wherein the agent orother treatment is or comprises tocilizumab.

191. The method of any of embodiments 169-190, wherein the agent orother treatment is or comprises siltuximab.

192. The method of embodiment 186, wherein the steroid is or comprisesdexamethasone.

193. The method of embodiment 186, wherein the agent capable ofpreventing, blocking or reducing microglial cell activity or function isselected from an anti-inflammatory agent, an inhibitor of NADPH oxidase(NOX2), a calcium channel blocker, a sodium channel blocker, inhibitsGM-CSF, inhibits CSF1R, specifically binds CSF-1, specifically bindsIL-34, inhibits the activation of nuclear factor kappa B (NF-κB),activates a CB₂ receptor and/or is a CB₂ agonist, a phosphodiesteraseinhibitor, inhibits microRNA-155 (miR-155) or upregulates microRNA-124(miR-124).

194. The method of embodiment 193, wherein the agent capable ofpreventing, blocking or reducing microglial cell activation or functionis a small molecule, peptide, protein, antibody or antigen-bindingfragment thereof, an antibody mimetic, an aptamer, or a nucleic acidmolecule.

195. The method of embodiment 193 or embodiment 194, wherein the agentis selected from minocycline, naloxone, nimodipine, Riluzole, MOR103,lenalidomide, a cannabinoid (optionally WIN55 or 212-2), intravenousimmunoglobulin (IVIg), ibudilast, anti-miR-155 locked nucleic acid(LNA), MCS110, PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527,JNJ28312141, ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945, emactuzumab, IMC-CS4, FPA008,LY-3022855, AMG-820 and TG-3003.

196. The method of any of embodiments 193-195, wherein the agent is aninhibitor of colony stimulating factor 1 receptor (CSF1R).

197. The method of any of embodiments 193-196, wherein the agent isselected from:

PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141,ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945 or a pharmaceutical salt or prodrugthereof;

emactuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003 or is anantigen-binding fragment thereof; or a combination of any of theforegoing.

198. The method of any of embodiments 193-197, wherein the inhibitor isPLX-3397.

199. The method of any of embodiments 1-23 and 109-198, wherein therecombinant receptor specifically binds to an antigen associated withthe disease or condition or expressed in cells of the environment of alesion associated with the disease or condition.

200. The method of any of embodiments 1-23 and 109-199, wherein thedisease or condition is a cancer.

201. The method of any of embodiments 1-23 and 109-200, wherein thedisease or condition is a myeloma, leukemia or lymphoma.

202. The method of any of embodiments 1-23 and 109-201, wherein thedisease or condition is a B cell malignancy and/or is acutelymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia(CLL), non-Hodgkin lymphoma (NHL), and Diffuse Large B-Cell Lymphoma(DLBCL).

203. The method of any of embodiments 1-23 and 109-202, wherein theantigen is ROR1, B cell maturation antigen (BCMA), carbonic anhydrase 9(CAIX), tEGFR, Her2/neu (receptor tyrosine kinase erbB2), L1-CAM, CD19,CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen,anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR,epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40),EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate bindingprotein (FBP), FCRLS, FCRHS, fetal acetylcholine receptor, GD2, GD3,HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor(kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM),Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentiallyexpressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AIMAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8,avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2Dligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin,murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100,oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA),Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), acyclin, cyclin A2, CCL-1, CD138, G Protein Coupled Receptor 5D (GPCRSD),or a pathogen-specific antigen.

204. The method of any of embodiments 1-23 and 109-203, wherein therecombinant receptor is a T cell receptor or a functional non-T cellreceptor.

205. The method of any of embodiments 1-23 and 109-204, wherein therecombinant receptor is a chimeric antigen receptor (CAR).

206. The method of embodiment 205, wherein the CAR comprises anextracellular antigen-recognition domain that specifically binds to theantigen and an intracellular signaling domain comprising an ITAM,wherein optionally, the intracellular signaling domain comprises anintracellular domain of a CD3-zeta (CD3ζ) chain; and/or wherein the CARfurther comprises a costimulatory signaling region, which optionallycomprises a signaling domain of CD28 or 4-1BB.

207. The method of any of embodiments 1-23 and 109-206, wherein theengineered cells comprise T cells, optionally CD4⁺ and/or CD8⁺.

208. The method of embodiment 207, wherein the T cells are primary Tcells obtained from a subject.

209. The method of any of embodiments 1-23 and 109-208, wherein the celltherapy comprises the administration of from or from about 1×10⁵ to1×10⁸ total recombinant receptor-expressing cells, total T cells, ortotal peripheral blood mononuclear cells (PBMCs), from or from about5×10⁵ to 1×10⁷ total recombinant receptor-expressing cells, total Tcells, or total peripheral blood mononuclear cells (PBMCs) or from orfrom about 1×10⁶ to 1×10⁷ total recombinant receptor-expressing cells,total T cells, or total peripheral blood mononuclear cells (PBMCs), eachinclusive.

210. The method of any of embodiments 1-23 and 109-209, wherein the celltherapy comprises the administration of no more than 1×10⁸ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), no more than 1×10⁷ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), no more than 0.5×10⁷ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), no more than 1×10⁶ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs), no more than 0.5×10⁶ totalrecombinant receptor-expressing cells, total T cells, or totalperipheral blood mononuclear cells (PBMCs).

211. The method of any of embodiments 109-210, wherein the dose that isnot associated with risk of developing toxicity or severe toxicity is orcomprises less than or less than about 5×10⁷ total recombinantreceptor-expressing cells, optionally CAR⁺ cells, total T cells, ortotal peripheral blood mononuclear cells (PBMCs), such as less than orless than about 2.5×10⁷, less than or less than about 1.0×10⁷, less thanor less than about 5.0×10⁶, less than or less than about 1.0×10⁶, lessthan or less than about 5.0×10⁵, or less than or less than about 1×10⁵total recombinant receptor-expressing cells, optionally CAR⁺ cells,total T cells, or total peripheral blood mononuclear cells (PBMCs).

212. The method of any of embodiments 109-211, wherein the dose that isnot associated with risk of developing toxicity or severe toxicity is orcomprises from or from about 1×10⁵ to 5×10⁷ total recombinantreceptor-expressing cells, optionally CAR⁺ cells, total T cells, ortotal peripheral blood mononuclear cells (PBMCs), such as 1×10⁵ to2.5×10⁷, 1×10⁵ to 1.0×10⁷, 1×10⁵ to 5.0×10⁶, 1×10⁵ to 1.0×10⁶, 1.0×10⁵to 5.0×10⁵, 5.0×10⁵ to 5×10⁷, 5×10⁵ to 2.5×10⁷, 5×10⁵ to 1.0×10⁷, 5×10⁵to 5.0×10⁶, 5×10⁵ to 1.0×10⁶, 1.0×10⁶ to 5×10⁷, 1×10⁶ to 2.5×10⁷, 1×10⁶to 1.0×10⁷, 1×10⁶ to 5.0×10⁶, 5.0×10⁶ to 5×10⁷, 5×10⁶ to 2.5×10⁷, 5×10⁶to 1.0×10⁷, 1.0×10⁷ to 5×10⁷, 1×10⁷ to 2.5×10⁷ or 2.5×10⁷ to 5×10⁷ totalrecombinant receptor-expressing cells, optionally CAR⁺ cells, total Tcells, or total peripheral blood mononuclear cells (PBMCs).

213. The method of any of embodiments 109-212, wherein the engineeredcells are autologous to the subject.

214. The method of any of embodiments 109-213, wherein the engineeredcells are allogeneic to the subject.

215. The method of any of embodiments 109-214, wherein the reagent isdetectably labeled, optionally fluorescently labeled.

216. The method of any of embodiments 163-215, wherein the one or moreanalyte is LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-alpha, IFN-alpha2,MCP-1 and MCP-1beta.

217. The method of any of embodiments 163-215, wherein the one or moreanalyte is selected from LDH, ferritin, CRP, IL-6, IL-8, IL-10, TNF-α,MCP-1 and MIP-1beta, and the toxicity is neurotoxicity.

218. The method of any of embodiments 163-215, wherein the one or moreanalyte is selected from IL-8 and IL-10 and the toxicity isneurotoxicity, optionally severe neurotoxicity or grade 3 or higherneurotoxicity.

219. The method of any of embodiments 163-215, wherein the one or moreanalyte is selected from among LDH, Ferritin, C-reactive protein (CRP),D-dimer (fibrin degradation product), IL-6, IL-10, IL-15, IL-16 TNF-α,MIP-1α and MIP-1β and the toxicity is CRS or neurotoxicity.

220. The method of any of embodiments 109-219, wherein the one or moreanalyte is or comprises LDH.

221. An article of manufacture comprising one or more dose of a celltherapy, each dose comprising cells expressing a chimeric antigenreceptor (CAR), and instructions for administering the cell therapy,wherein the instructions specify that:

the dose of cells is to be administered to a subject having oridentified to have non-Hodgkin lymphoma (NHL), the NHL selected fromdiffuse large B cell lymphoma (DLBCL), primary mediastinal large B celllymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma),or follicular lymphoma Grade 3B, wherein the subject is or has beenidentified as having an Eastern Cooperative Oncology Group PerformanceStatus (ECOG) status of 0 or 1; and

the instructions specify administration of a number of T cells or ofcells of a specified phenotype, or specify administration of an amountor volume of one or more formulations corresponding to or containingsaid specified number of cells, wherein the specified number of cellscomprises between at or about 5×10⁷ CAR-expressing T cells and 1×10⁸CAR-expressing T cells, inclusive, between at or about 5×10⁷CAR-expressing T cells and at or about 1.5×10⁸ CAR-expressing T cells,at or about 5×10⁷ CAR-expressing T cells, at or about 1×10⁸CAR-expressing T cells, or at or about 1.5×10⁸ CAR-expressing T cells,between at or about 2.5×10⁷ CD8⁺CAR-expressing T cells and at or about5×10⁷ CD8⁺ CAR-expressing T cells, inclusive, between at or about2.5×10⁷ CAR-expressing T cells and at or about 0.75×10⁸ CD8⁺CAR-expressing T cells, or at or about 2.5×10⁷ CD8⁺ CAR-expressing Tcells, at or about 5×10⁷ CAR-expressing T cells, or at or about 0.75×10⁸CD8⁺ CAR-expressing T cells, or of viable populations of any of theforegoing.

222. The article of manufacture of embodiment 221, wherein theinstructions specify administering the cell therapy at a defined ratioof CD4⁺ cells expressing the CAR to CD8⁺ cells, or specify administeringamounts of volumes of the formulation(s) corresponding to such definedratio, or comprises a formulation having the cells at such ratio orcomprises the cells at such ratio expressing the CAR and/or of CD4⁺cells to CD8⁺ cells, which ratio optionally is approximately 1:1 or isbetween approximately 1:3 and approximately 3:1.

223. An article of manufacture comprising a cell therapy, or one of aplurality of compositions of a cell therapy, comprising a dose orcomposition of genetically engineered cells expressing a chimericantigen receptor (CAR), and instructions for administering the celltherapy, wherein the instructions specify:

the dose of T cells is to be administered at a defined ratio of CD4⁺cells expressing the CAR to CD8⁺ cells expressing the CAR and/or of CD4⁺cells to CD8⁺ cells, which ratio is approximately or is 1:1; and

the dose of cells is to be administered to a subject having oridentified to have non-Hodgkin lymphoma (NHL), the NHL selected fromdiffuse large B cell lymphoma (DLBCL), primary mediastinal large B celllymphoma (PMBCL), NOS (de novo or transformed from indolent lymphoma),or follicular lymphoma Grade 3B.

224. An article of manufacture comprising a cell therapy, or one of aplurality of compositions of a cell therapy, comprising a dose orcomposition of genetically engineered cells expressing a chimericantigen receptor (CAR), and instructions for administering the celltherapy, wherein the instructions specify:

the dose of cells is to be administered to a subject having oridentified to have non-Hodgkin lymphoma (NHL), optionally an NHLselected from aggressive NHL, diffuse large B cell lymphoma (DLBCL), NOS(de novo and transformed from indolent), primary mediastinal large Bcell lymphoma (PMBCL), mantle cell lymphoma (MCL), and/or follicularlymphoma (FL), optionally follicular lymphoma Grade 3B (FL3B);

the dose of T cells to be administered comprises between at or about5×10⁷ CAR-expressing T cells and 1×10⁸ CAR-expressing T cells,inclusive; and

the dose of T cells is to be administered at a defined ratio of CD4⁺cells expressing the CAR to CD8⁺ cells expressing the CAR and/or of CD4⁺cells to CD8⁺ cells, which ratio is approximately or is 1:1.

225. The article of manufacture of embodiment 222 or embodiment 223,wherein the instructions further specify the cell therapy is to beadministered to a subject that is or has been identified as having anEastern Cooperative Oncology Group Performance Status (ECOG) status of0, 1 or 2 and/or that is or has been identified as having an ECOG statusof 0 or 1.

226. The article of manufacture of any of embodiments 221-225, wherein:

the instructions specify that the administration is to a subject thathas not received, immediately prior to the administration of the dose ofcells or within or about 1 month of the dose of cells, an agent ortreatment capable of treating, preventing, delaying, reducing orattenuating the development or risk of development of a toxicity; and/or

the instructions do not specify that the administration to a subjectwho, prior to initiation of administration of the dose of cells, hasbeen administered an anti-IL-6 or anti-IL-6R antibody, optionallytocilizumab or siltuximab, and/or has not been administered a steroid,optionally dexamethasone; and/or

the instructions do not specify administering an agent that is ananti-IL-6 or anti-IL-6R antibody, optionally tocilizumab or siltuximab,a steroid, optionally dexamethasone, and/or an agent for the purpose ofprophylactically reducing the risk of or treating CRS or neurotoxicity,and/or do not specify administering said agent within a period of timefollowing administration of the dose of cells, which period of time isoptionally at or about 1, 2, 3, 4, 5 days, or is optionally at or about6, 7, 8, 9, 10, 11 days, or is optionally 1, 2, 3 or 4 weeks, and/or donot specify administering said agent prior to the subject exhibiting, orunless the subject exhibits, a sign or symptom of neurotoxicity and/orCRS, optionally other than a fever, optionally wherein the fever is nota sustained fever or the fever is or has been reduced or reduced by morethan 1° C. after treatment with an antipyretic; and/or

the instructions specify administration of the cell dose on anoutpatient basis and/or without admitting the subject to a hospitaland/or without an overnight stay at a hospital and/or without requiringadmission to or an overnight stay at a hospital, optionally unless arisk factor specific to the subject or cells of the subject is otherwiseidentified in the instructions or information in the article oroptionally unless or until the subject exhibits a sustained fever or afever that is or has not been reduced or not reduced by more than 1° C.after treatment with an antipyretic.

227. The article of manufacture of embodiment 226, wherein theinstructions further specify that if, after administration of the celldose on an outpatient basis without requiring admission to or anovernight stay at a hospital, the subject exhibits a sustained fever ora fever that is or has not been reduced or not reduced by more than 1°C. after treatment with an antipyretic, the subject is to be admitted tothe hospital or to an overnight stay at a hospital and/or is to beadministered an agent or treatment for the treatment or prevention orreduction or attenuation of a neurotoxicity and/or a cytokine releasesyndrome or risk thereof.

228. The article of manufacture of embodiment 226 or embodiment 227,wherein the agent is or comprises an anti-IL-6 or anti-IL-6R antibody,optionally tocilizumab or siltuximab, and/or a steroid, optionallydexamethasone.

229. The article of manufacture of any of embodiments 221-228, whereinthe instructions specify the subject has a DLBCL characterized as denovo or transformed from an indolent disease and/or does not have aDLBCL transformed from MZL and CLL (Richter's).

230. The article of manufacture of any of embodiments 221-229, whereinthe instructions specify the subject does not have primary mediastinallarge B cell lymphoma (PMBCL).

231. The article of manufacture of any of embodiments 221-230, whereinthe instruction specify the administration of the cells is in a subjectthat is or has been identified as having a lymphoma associated with orinvolving central nervous system (CNS) involvement.

232. The article of manufacture of any of embodiments 221-231, whereinthe instructions specify the administration of the cell therapy is for asubject that is or has been identified as having a double/triple hitlymphoma, is or has been identified as having a chemorefractorylymphoma, optionally a chemorefractory DLBCL; that has not achievedcomplete remission (CR) in response to a prior therapy; and/or hasrelapsed within 1 year or less than 1 year after receiving an autologousstem cell transplant (ASCT).

233. The article of manufacture of any of embodiments 221-232, whereinthe instructions specify further administering to a subject anadditional therapeutic agent or therapy, optionally other than a celltherapy, optionally other than a CAR⁺ T cell therapy.

234. The article of manufacture of embodiment 233, wherein theadditional therapeutic agent or therapy is an agent for treating the NHLor malignancy and/or increases the persistence, activity and/or efficacyof the dose of cells.

235. The article of manufacture of embodiment 233 or embodiment 234,wherein the instructions specify administration of the additionaltherapeutic agent or therapy is in a subject that does not exhibit aresponse, optionally does not exhibit a CR or OR, to the cell therapywithin 1 month, within 2 months or within 3 months after administrationof the dose of cells.

236. The article of manufacture of any of embodiments 233-235, whereinthe instructions specify the administration of the additionaltherapeutic agent or therapy is in a subject:

that is or has been identified to have stable or progressive disease(SD/PD) following treatment with a prior therapy, optionally a priortherapy with a chemotherapeutic agent;

that is or has been identified with an Eastern Cooperative OncologyGroup Performance Status (ECOG) status of 2;

that is or has been identified as having a transformed follicularlymphoma (tFL); or

that is or has been identified has having a DLBCL transformed from MZLand CLL.

237. The article of manufacture of any of embodiments 233-236, whereinthe instructions specify the additional therapeutic agent or therapy isfor administration prior to, with or at the same time and/or subsequentto initiation of administration of the dose of cells.

238. The article of manufacture of any of embodiments 221-237, whereinthe CAR comprises an scFv specific for the antigen, a transmembranedomain, a cytoplasmic signaling domain derived from a costimulatorymolecule, which optionally is a 4-1BB, and a cytoplasmic signalingdomain derived from a primary signaling ITAM-containing molecule, whichoptionally is a CD3zeta.

239. The article of manufacture of any of embodiments 221-238, whereinthe antigen is a B cell antigen, which optionally is CD19.

240. The article of manufacture of any of embodiments 221-239, furthercomprising instructions for use with, after or in connection with alymphodepleting therapy, the lymphdepleting therapy optionallycomprising fludarabine and/or cyclophosphamide.

241. The article of manufacture of embodiment 240, wherein thelymphodepleting therapy comprises administration of cyclophosphamide atabout 200-400 mg/m², optionally at or about 300 mg/m², inclusive, and/orfludarabine at about 20-40 mg/m², optionally 30 mg/m², daily for 2-4days, optionally for 3 days.

242. The article of manufacture of embodiment 240 or embodiment 241,wherein the lymphodepleting therapy comprises administration ofcyclophosphamide at or about 300 mg/m² and fludarabine at about 30mg/m²daily for 3 days.

243. The article of manufacture of any of embodiments 221-242, whereinthe instructions further specify the administration of the cell therapyis to be or may be administered to the subject on an outpatient settingand/or without admission of the subject to the hospital overnight or forone or more consecutive days and/or is without admission of the subjectto the hospital for one or more days, optionally unless or until thesubject exhibits a sustained fever or a fever that is or has not beenreduced or not reduced by more than 1° C. after treatment with anantipyretic.

244. The article of manufacture of embodiment 243, wherein if thesubject exhibits a sustained fever or a fever that is or has not beenreduced or not reduced by more than 1° C. after treatment with anantipyretic, the instructions further specify the subject is to beadmitted to the hospital or to an overnight stay at a hospital and/or isto be administered an agent or treatment for the treatment or preventionor reduction or attenuation of a neurotoxicity and/or a cytokine releasesyndrome or risk thereof.

245. The article of manufacture of any of embodiments 221-244, whereinthe instructions further specify the cell therapy is for parenteraladministration, optionally intravenous administration.

246. The article of manufacture of any of embodiments 221-245, whereinthe cell therapy comprises primary T cells obtained from a subject.

247. The article of manufacture of any of embodiments 221-246, whereinthe T cells are autologous to the subject.

248. The article of manufacture of any of embodiments 221-246, whereinthe T cells are allogeneic to the subject.

249. The article of manufacture of any of embodiments 221-248, whereinthe article of manufacture comprises one of a plurality of compositionsof the cell therapy comprising a first composition of geneticallyengineered cells comprising CD4⁺ T cells or CD8⁺ T cells, wherein theinstructions specify the first composition is for use in with a secondcomposition comprising the other of the CD4⁺ T cells or the CD8⁺ Tcells, optionally wherein the cells of the first composition and cellsof the same composition are from the same subject.

250. The article of manufacture of embodiment 249, wherein theinstructions specify the first composition and second composition are tobe administered at a defined ratio of CD4⁺ cells expressing therecombinant receptor to CD8⁺ cells expressing the recombinant receptorand/or of CD4⁺ cells to CD8⁺ cells, which ratio optionally isapproximately 1:1 or is between approximately 1:3 and approximately 3:1.

251. The article of manufacture of embodiment 250, wherein the definedratio is or is approximately 1:1.

252. The article of manufacture of any of embodiments 221-251, whereinthe composition further comprises a cryoprotectant and/or the articlefurther includes instructions for thawing the composition prior toadministration to the subject.

253. The article of manufacture of any of embodiments 249-252, whereinthe instructions specify administering the composition comprising theCD4⁺ T cells and the composition comprising the CD8⁺ T cells 0 to 12hours apart, 0 to 6 hours apart or 0 to 2 hours apart.

254. The article of manufacture of any of embodiments 249-253, whereinthe instructions specify administering the composition comprising theCD4⁺ T cells and the composition comprising the CD8⁺ T cells no morethan 2 hours, no more than 1 hour, no more than 30 minutes, no more than15 minutes, no more than 10 minutes or no more than 5 minutes apart.

255. The article of manufacture of any of embodiments 249-254, whereinthe instructions specify administering the composition comprising theCD4⁺ T cells prior to administering the composition comprising the CD8⁺cells.

256. The article of manufacture of any of embodiments 249-254, whereinthe instructions specify administering the composition comprising theCD8⁺ T cells prior to administering the composition comprising the CD4⁺cells.

257. An article of manufacture comprising one or more reagent capable ofdetecting one or more analytes, and instructions for using the reagentto assay a biological sample from a subject that is a candidate fortreatment, optionally with a cell therapy, said cell therapy optionallycomprising a dose or composition of genetically engineered cellsexpressing a recombinant receptor, wherein the one or more analytes isselected from LDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16,TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha,IL-1Rbeta, IP-10, perforin and D-dimer (fibrin degradation product).

258. The article of manufacture of embodiment 257, further comprisingthe cell therapy and/or further comprising instructions for use with,prior to and/or in connection with treatment with the cell therapy.

259. The article of manufacture of embodiment 257 or embodiment 258,further comprising one or more agents or treatments for treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity and/or instructions for the administration ofone or more agents or treatments for treating, preventing, delaying,reducing or attenuating the development or risk of development of atoxicity in the subject.

260. The article of manufacture of any of embodiments 257-259, whereinthe instructions further specify, if the level, amount or concentrationof the analyte in the sample is at or above a threshold level for theanalyte:

administering to the subject an agent or other treatment capable oftreating, preventing, delaying, reducing or attenuating the developmentor risk of development of a toxicity (i) prior to, (ii) within one, two,or three days of, (iii) concurrently with and/or (iv) at first feverfollowing, the initiation of administration of the cell therapy to thesubject; and/or

administering to the subject the cell therapy at a reduced dose or at adose that is not associated with risk of developing toxicity or severetoxicity, or is not associated with a risk of developing a toxicity orsevere toxicity in a majority of subjects, and/or a majority of subjectshaving a disease or condition that the subject has or is suspected ofhaving, following administration of the cell therapy; and/or

administering to the subject the cell therapy in an in-patient settingand/or with admission to the hospital for one or more days, optionallywherein the cell therapy is otherwise to be administered to subjects onan outpatient basis or without admission to the hospital for one or moredays.

261. The article of manufacture of any of embodiments 257-259, whereinthe instructions further specify, if the level, amount or concentrationof the analyte is below a threshold level for the analyte, administeringto the subject the cell therapy, optionally at a non-reduced dose,optionally on an outpatient basis or without admission to the hospitalfor one or more days.

262. The article of manufacture of any of embodiments 257-261, whereinthe instructions further specify administering the cell therapy to thesubject and wherein the instructions further specify, if the level,amount or concentration of the analyte, is below a threshold level:

the administration of the cell therapy does not comprise administering,prior to or concurrently with administering the cell therapy and/orprior to the development of a sign of symptom of a toxicity other thanfever, an agent or treatment capable of treating, preventing, delaying,or attenuating the development of the toxicity; and/or

the administration of the cell therapy is to be or may be administeredto the subject on an outpatient setting and/or without admission of thesubject to the hospital overnight or for one or more consecutive daysand/or is without admission of the subject to the hospital for one ormore days.

263. The article of manufacture of any of embodiments 257-262, whereinthe threshold level is within 25%, within 20%, within 15%, within 10% orwithin 5% of the average level, amount or concentration, and/or iswithin a standard deviation of the average level, amount orconcentration, of the analyte in a biological sample obtained from agroup of subjects prior to receiving a recombinant receptor-expressingtherapeutic cell composition, wherein each of the subjects of the groupwent on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

264. An article of manufacture comprising:

a cell therapy, said cell therapy optionally comprising a dose orcomposition of genetically engineered cells expressing a recombinantreceptor, and

instructions for administering the cell therapy following or based onthe results of an assessment, in a biological sample of the level, oramount or concentration of one or more analyte in a biological sample,said biological sample obtained from the subject prior to administeringthe cell therapy and/or said biological sample not comprising therecombinant receptor and/or said engineered cells, wherein the one ormore analytes is selected from LDH, ferritin, CRP, IL-6, IL-7, IL-8,IL-10, IL-15, IL-16, TNF-alpha, IFN-gamma, MCP-1, MIP-1beta, eotaxin,G-CSF, IL-1Ralpha, IL-1Rbeta, IP-10, perforin, and D-dimer (fibrindegradation product).

265. The article of manufacture of embodiment 264, wherein saidassessment comprises detection which optionally comprises contacting areagent capable of directly or indirectly detecting the analyte with thebiological sample and determining the level, amount or concentration ofthe analyte in the biological sample.

266. The article of manufacture of embodiment 265, further comprisingthe reagent and/or further comprising instructions for use with, priorto and/or in connection with the reagent for detecting the analyte.

267. The article of manufacture of any of embodiments 264-266, furthercomprising one or more agents or treatments for treating, preventing,delaying, reducing or attenuating the development or a risk ofdevelopment of a toxicity and/or instructions for the administration ofone or more agents or treatments for treating, preventing, delaying,reducing or attenuating the development or risk of development of atoxicity in the subject.

268. The article of manufacture of any of embodiments 264-267, whereinthe instructions for administering the cell therapy specify, if thelevel, amount or concentration of the analyte in the sample, is at orabove a threshold level:

administering to the subject an agent or other treatment capable oftreating, preventing, delaying, reducing or attenuating the developmentor risk of development of a toxicity (i) prior to, (ii) within one, two,or three days of, (iii) concurrently with and/or (iv) at first feverfollowing, the initiation of administration of administration of thetherapeutic cell composition or the genetically engineered cells; and/or

administering to the subject the cell therapy at a reduced dose or at adose that is not associated with risk of developing toxicity or severetoxicity, or is not associated with a risk of developing a toxicity orsevere toxicity in a majority of subjects, and/or a majority of subjectshaving a disease or condition that the subject has or is suspected ofhaving, following administration of the cell therapy; and/or

administering to the subject the cell therapy in an in-patient settingand/or with admission to the hospital for one or more days, optionallywherein the cell therapy is otherwise to be administered to subjects onan outpatient basis or without admission to the hospital for one or moredays.

269. The article of manufacture of any of embodiments 264-268, whereinthe instructions for administering the cell therapy specify, if thelevel, amount or concentration of the analyte in the sample, is below athreshold level, administering to the subject the cell therapy,optionally at a non-reduced dose, optionally on an outpatient basis orwithout admission to the hospital for one or more days.

270. The article of manufacture of any of embodiments 264-269, whereinthe instructions further specify administering the cell therapy to thesubject and wherein the instructions further specify, if the level,amount or concentration of the analyte is below a threshold level:

not administering, prior to or concurrently with administering the celltherapy and/or prior to the development of a sign or symptom of atoxicity other than fever, an agent or treatment capable of treating,preventing, delaying, or attenuating the development of the toxicity;and/or

the administration of the cell therapy is to be or may be administeredto the subject on an outpatient setting and/or without admission of thesubject to the hospital overnight or for one or more consecutive daysand/or is without admission of the subject to the hospital for one ormore days.

271. The article of manufacture of any of embodiments 264-270, whereinthe threshold level is within 25%, within 20%, within 15%, within 10% orwithin 5% of the average level, amount or concentration, and/or iswithin a standard deviation of the average level, amount orconcentration, of the analyte in a biological sample obtained from agroup of subjects prior to receiving a recombinant receptor-expressingtherapeutic cell composition, wherein each of the subjects of the groupwent on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

272. An article of manufacture comprising an agent capable of treating,preventing, delaying, reducing or attenuating the development or risk ofdevelopment of a toxicity, and instructions for administering the agentfollowing or based on the results of an assessment in a biologicalsample of the level, amount or concentration of one or more analytes ina biological sample, wherein the one or more analytes is selected fromLDH, ferritin, CRP, IL-6, IL-7, IL-8, IL-10, IL-15, IL-16, TNF-alpha,IFN-gamma, MCP-1, MIP-1beta, eotaxin, G-CSF, IL-1Ralpha, IL-1Rbeta,IP-10, perforin, and D-dimer (fibrin degradation product).

273. The article of manufacture of embodiment 272, wherein saidassessment comprises detection which optionally comprises contacting areagent capable of directly or indirectly detecting the analyte with thebiological sample and determining the level, amount or concentration ofthe analyte in the biological sample.

274. The article of manufacture of embodiment 272 or embodiment 273,wherein the instructions specify that the agent is to be administered i)prior to, (ii) within one, two, or three days of, (iii) concurrentlywith and/or (iv) at first fever following, the initiation ofadministration of the cell therapy to the subject and/or furthercomprises instructions for use with, prior to and/or in connection withtreatment with the cell therapy.

275. The article of manufacture of any of embodiments 272-274, whereinsaid biological sample is obtained from the subject prior toadministering the agent or cell therapy.

276. The article of manufacture of any of embodiments 257-275, whereinthe reagent is a binding molecule that specifically binds to theanalyte.

277. The article of manufacture of any of embodiments 257-276, whereinthe reagent is an antibody or an antigen-binding fragment thereof.

278. The article of manufacture of any of embodiments 257-277, whereinthe biological sample is or is obtained from a blood, plasma or serumsample.

279. The article of manufacture of any of embodiments 272-278, furthercomprising the reagent for detecting the analyte and/or furthercomprising instructions for use with, prior to and/or in connection withthe reagent for detecting the analyte.

280. The article of manufacture of any of embodiments 272-279, furthercomprising the cell therapy and/or further comprising instructions foruse with, prior to and/or in connection with treatment with the celltherapy.

281. The article of manufacture of any of embodiments 272-280, whereinthe instructions for administering the agent specify, if the level,amount or concentration of the analyte in the sample, is at or above athreshold level administering to the subject the agent.

282. The article of manufacture of embodiment 281, wherein theinstruction further specify administering a cell therapy to the subject,wherein administration of the agent is to be carried out (i) prior to,(ii) within one, two, or three days of, (iii) concurrently with and/or(iv) at first fever following, the initiation of administration of thecell therapy to the subject.

283. The article of manufacture of any of embodiments 272-282, whereinthe instructions for administering the agent specify, if the level,amount or concentration is below the threshold level administering tothe subject the cell therapy, optionally wherein the instructionsspecify the cell therapy is to be or may be administered to the subjecton an outpatient setting and/or without admission of the subject to thehospital overnight or for one or more consecutive days and/or is withoutadmission of the subject to the hospital for one or more days.

284. The article of manufacture of any of embodiments 272-283, whereinthe threshold level is within 25%, within 20%, within 15%, within 10% orwithin 5% of the average level, amount or concentration, and/or iswithin a standard deviation of the average level, amount orconcentration, of the analyte in a biological sample obtained from agroup of subjects prior to receiving a recombinant receptor-expressingtherapeutic cell composition, wherein each of the subjects of the groupwent on to develop a toxicity after receiving arecombinant-receptor-expressing therapeutic cell composition fortreating the same disease or condition.

285. The article of manufacture of any of embodiments 257-284, whereinassaying or assessing cells for the analyte is by an immunoassay.

286. The article of manufacture of any of embodiments 257-285, whereinthe toxicity comprises neurotoxicity or cytokine release syndrome (CRS),optionally grade 1 or higher neurotoxicity or CRS.

287. The article of manufacture of any of embodiments 257-286, wherein:

the toxicity comprises severe neurotoxicity and/or comprises a grade 2or higher neurotoxicity, a grade 3 or higher neurotoxicity, at leastprolonged grade 3 neurotoxicity or is at or above grade 4 or grade 5neurotoxicity; and/or

the toxicity comprises severe CRS and/or comprises grade 2 or higher orgrade 3 or higher CRS.

288. The article of manufacture of any of embodiments 257-287, whereinthe toxicity is associated with cerebral edema.

289. The article of manufacture of any of embodiments 257-288, whereinthe agent or other treatment is or comprises one or more of a steroid;an antagonist or inhibitor of a cytokine receptor or cytokine selectedfrom among IL-10, IL-10R, IL-6, IL-6 receptor, IFNγ, IFNGR, IL-2,IL-2R/CD25, MCP-1, CCR2, CCR4, MIP1β, CCR5, TNFalpha, TNFR1, IL-1, andIL-1Ralpha/IL-1beta; or an agent capable of preventing, blocking orreducing microglial cell activity or function.

290. The article of manufacture of embodiment 289, wherein theantagonist or inhibitor is or comprises an agent selected from among anantibody or antigen-binding fragment, a small molecule, a protein orpeptide and a nucleic acid.

291. The article of manufacture of any of embodiments 257-290, whereinthe agent or other treatment is an anti-IL-6 antibody or an anti-IL6receptor antibody.

292. The article of manufacture of any of embodiments 257-291, whereinthe agent or other treatment is or comprises an agent selected fromamong tocilizumab, siltuximab, clazakizumab, sarilumab, olokizumab(CDP6038), elsilimomab, ALD518/BMS-945429, sirukumab (CNTO 136),CPSI-2634, ARGX-109, FE301 and FM101.

293. The article of manufacture of any of embodiments 257-292, whereinthe agent or other treatment is or comprises tocilizumab.

294. The article of manufacture of any of embodiments 257-293, whereinthe agent or other treatment is or comprises siltuximab.

295. The article of manufacture of embodiment 289, wherein the steroidis or comprises dexamethasone.

296. The article of manufacture of embodiment 289, wherein the agentcapable of preventing, blocking or reducing microglial cell activity orfunction is selected from an anti-inflammatory agent, an inhibitor ofNADPH oxidase (NOX2), a calcium channel blocker, a sodium channelblocker, inhibits GM-CSF, inhibits CSF1R, specifically binds CSF-1,specifically binds IL-34, inhibits the activation of nuclear factorkappa B (NF-κB), activates a CB₂ receptor and/or is a CB₂ agonist, aphosphodiesterase inhibitor, inhibits microRNA-155 (miR-155) orupregulates microRNA-124 (miR-124).

297. The article of manufacture of embodiment 296, wherein the agentcapable of preventing, blocking or reducing microglial cell activationor function is a small molecule, peptide, protein, antibody orantigen-binding fragment thereof, an antibody mimetic, an aptamer, or anucleic acid molecule.

298. The article of manufacture of embodiment 296 or embodiment 297wherein the agent is selected from minocycline, naloxone, nimodipine,Riluzole, MOR103, lenalidomide, a cannabinoid, optionally WIN55 or212-2, intravenous immunoglobulin (IVIg), ibudilast, anti-miR-155 lockednucleic acid (LNA), MCS110, PLX-3297, PLX647, PLX108-D1, PLX7486,JNJ-40346527, JNJ28312141, ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945, emactuzumab, IMC-CS4, FPA008,LY-3022855, AMG-820 and TG-3003.

299. The article of manufacture of any of embodiments 296-298, whereinthe agent is an inhibitor of colony stimulating factor 1 receptor(CSF1R).

300. The article of manufacture of any of embodiments 296-299, whereinthe inhibitor is selected from:

PLX-3397, PLX647, PLX108-D1, PLX7486, JNJ-40346527, JNJ28312141,ARRY-382, AC-708, DCC-3014,5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine(GW2580), AZD6495, Ki20227, BLZ945 or a pharmaceutical salt or prodrugthereof;

emactuzumab, IMC-CS4, FPA008, LY-3022855, AMG-820 and TG-3003 or is anantigen-binding fragment thereof; or a combination of any of theforegoing.

301. The article of manufacture of any of embodiments 296-300, whereinthe inhibitor is PLX-3397.

302. The article of manufacture of any of embodiments 24-48 and 257-301,wherein the recombinant receptor specifically binds to an antigenassociated with the disease or condition or expressed in cells of theenvironment of a lesion associated with the disease or condition.

303. The article of manufacture of any of embodiments 24-48 and 257-302,wherein the disease or condition is a cancer.

304. The article of manufacture of any of embodiments 24-48 and 257-303,wherein the disease or condition is a myeloma, leukemia or lymphoma.

305. The article of manufacture of any of embodiments 24-48 and 257-304,wherein the disease or condition is a B cell malignancy and/or is acutelymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia(CLL), non-Hodgkin lymphoma (NHL), and Diffuse Large B-Cell Lymphoma(DLBCL).

306. The article of manufacture of any of embodiments 302-305, whereinthe antigen is ROR1, B cell maturation antigen (BCMA), carbonicanhydrase 9 (CAIX), tEGFR, Her2/neu (receptor tyrosine kinase erbB2),L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surfaceantigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR,epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40),EPHa2, erb-B2, erb-B3, erb-B4, erbB dimers, EGFR vIII, folate bindingprotein (FBP), FCRLS, FCRHS, fetal acetylcholine receptor, GD2, GD3,HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insert domain receptor(kdr), kappa light chain, Lewis Y, L1-cell adhesion molecule, (L1-CAM),Melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, Preferentiallyexpressed antigen of melanoma (PRAME), survivin, TAG72, B7-H6, IL-13receptor alpha 2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AIMAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8,avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2Dligands, CD44v6, dual antigen, a cancer-testes antigen, mesothelin,murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY-ESO-1, MART-1, gp100,oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA),Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123,c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), acyclin, cyclin A2, CCL-1, CD138, G Protein Coupled Receptor 5D (GPCRSD),or a pathogen-specific antigen.

307. The article of manufacture of any of embodiments 24-48 and 257-306,wherein the recombinant receptor is a T cell receptor or a functionalnon-T cell receptor.

308. The article of manufacture of any of embodiments 24-48 and 257-307,wherein the recombinant receptor is a chimeric antigen receptor (CAR).

309. The article of manufacture of embodiment 308, wherein the CARcomprises an extracellular antigen-recognition domain that specificallybinds to the antigen and an intracellular signaling domain comprising anITAM, wherein optionally, the intracellular signaling domain comprisesan intracellular domain of a CD3-zeta (CD3ζ) chain; and/or wherein theCAR further comprises a costimulatory signaling region, which optionallycomprises a signaling domain of CD28 or 4-1BB.

310. The article of manufacture of any of embodiments 24-48 and 257-309,wherein the engineered cells comprise T cells, optionally CD4⁺ and/orCD8⁺.

311. The article of manufacture of embodiment 310, wherein the T cellsare primary T cells obtained from a subject.

312. The article of manufacture of any of embodiments 257-311, whereinthe dose that is not associated with risk of developing toxicity orsevere toxicity is or comprises less than or less than about 5×10⁷ totalrecombinant receptor-expressing cells, optionally CAR⁺ cells, total Tcells, or total peripheral blood mononuclear cells (PBMCs), such as lessthan or less than about 2.5×10⁷, less than or less than about 1.0×10⁷,less than or less than about 5.0×10⁶, less than or less than about1.0×10⁶, less than or less than about 5.0×10⁵, or less than or less thanabout 1×10⁵ total recombinant receptor-expressing cells, optionally CAR⁺cells, total T cells, or total peripheral blood mononuclear cells(PBMCs).

313. The article of manufacture of any of embodiments 257-312, whereinthe dose that is not associated with risk of developing toxicity orsevere toxicity is or comprises from or from about 1×10⁵ to 5×10⁷ totalrecombinant receptor-expressing cells, optionally CAR⁺ cells, total Tcells, or total peripheral blood mononuclear cells (PBMCs), such as1×10⁵ to 2.5×10⁷, 1×10⁵ to 1.0×10⁷, 1×10⁵ to 5.0×10⁶, 1×10⁵ to 1.0×10⁶,1.0×10⁵ to 5.0×10⁵, 5.0×10⁵ to 5×10⁷, 5×10⁵ to 2.5×10⁷, 5×10⁵ to1.0×10⁷, 5×10⁵ to 5.0×10⁶, 5×10⁵ to 1.0×10⁶, 1.0×10⁶ to 5×10⁷, 1×10⁶ to2.5×10⁷, 1×10⁶ to 1.0×10⁷, 1×10⁶ to 5.0×10⁶, 5.0×10⁶ to 5×10⁷, 5×10⁶ to2.5×10⁷, 5×10⁶ to 1.0×10⁷, 1.0×10⁷ to 5×10⁷, 1×10⁷ to 2.5×10⁷ or 2.5×10⁷to 5×10⁷ total recombinant receptor-expressing cells, optionally CAR⁺cells, total T cells, or total peripheral blood mononuclear cells(PBMCs).

314. The article of manufacture of any of embodiments 257-313, whereinthe reagent is detectably labeled, optionally fluorescently labeled.

315. The article of manufacture of any of embodiments 257-314, whereinthe one or more analyte is LDH, ferritin, CRP, IL-6, IL-8, IL-10,TNF-alpha, IFN-alpha2, MCP-1 and MCP-1beta.

316. The article of manufacture of any of embodiments 257-315, whereinthe one or more analyte is or comprises LDH.

317. The article of manufacture of any of embodiments 221-316, wherein:

the CAR comprises an scFv specific for the antigen, a transmembranedomain, a cytoplasmic signaling domain derived from a costimulatorymolecule, which optionally is or comprises a 4-1BB, and a cytoplasmicsignaling domain derived from a primary signaling ITAM-containingmolecule, which optionally is or comprises a CD3zeta signaling domainand optionally further comprises a spacer between the transmembranedomain and the scFv;

the CAR comprises, in order, an scFv specific for the antigen, atransmembrane domain, a cytoplasmic signaling domain derived from acostimulatory molecule, which optionally is or comprises a 4-1BBsignaling domain, and a cytoplasmic signaling domain derived from aprimary signaling ITAM-containing molecule, which optionally is aCD3zeta signaling domain; or

the CAR comprises, in order, an scFv specific for the antigen, a spacer,a transmembrane domain, a cytoplasmic signaling domain derived from acostimulatory molecule, which optionally is a 4-1BB signaling domain,and a cytoplasmic signaling domain derived from a primary signalingITAM-containing molecule, which optionally is or comprises a CD3zetasignaling domain; and wherein:

the spacer is optionally a polypeptide spacer that (a) comprises orconsists of all or a portion of an immunoglobulin hinge or a modifiedversion thereof or comprises about 15 amino acids or less, and does notcomprise a CD28 extracellular region or a CD8 extracellular region, (b)comprises or consists of all or a portion of an immunoglobulin hinge,optionally an IgG4 hinge, or a modified version thereof and/or comprisesabout 15 amino acids or less, and does not comprise a CD28 extracellularregion or a CD8 extracellular region, or (c) is at or about 12 aminoacids in length and/or comprises or consists of all or a portion of animmunoglobulin hinge, optionally an IgG4, or a modified version thereof;or (d) has or consists of the sequence of SEQ ID NO: 1, a sequenceencoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32,SEQ ID NO: 33, SEQ ID NO: 34, or a variant of any of the foregoinghaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto, or (e) comprisesor consists of the formula X₁PPX₂P, where X₁ is glycine, cysteine orarginine and X₂ is cysteine or threonine; and/or

the costimulatory domain comprises SEQ ID NO: 12 or a variant thereofhaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity thereto; and/or

the primary signaling domain comprises SEQ ID NO: 13 or 14 or 15 havingat least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more sequence identity thereto; and/or

the scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), aCDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence ofGNTLPYTFG (SEQ ID NO: 37) and/or a CDRH1 sequence of DYGVS (SEQ ID NO:38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and/or aCDRH3 sequence of YAMDYWG (SEQ ID NO: 40) or wherein the scFv comprisesa variable heavy chain region of FMC63 and a variable light chain regionof FMC63 and/or a CDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, aCDRL3 sequence of FMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence ofFMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as orcompetes for binding with any of the foregoing, and optionally whereinthe scFv comprises, in order, a V_(H), a linker, optionally comprisingSEQ ID NO: 24, and a V_(L), and/or the scFv comprises a flexible linkerand/or comprises the amino acid sequence set forth as SEQ ID NO: 24.

318. The article of manufacture of any of claims 221-317, wherein theinstructions provide information about a threshold level, individuallyfor each of the one or more analytes, that is indicative of whether asubject is likely to exhibit a response to treatment with the celltherapy.

319. The article of manufacture of any of claims 221-317, wherein theinstructions provide information about a threshold level, individuallyfor each of the one or more analytes, that is indicative of whether asubject is likely to exhibit a durable response following administrationof the cell therapy.

320. The article of manufacture of any of claims 221-317, wherein theinstructions provide information about a threshold level, individuallyfor each of the one or more analytes, that is indicative of whether asubject is likely to exhibit a toxicity following administration of thecell therapy.

VIII. Definitions

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues, and are not limited to a minimumlength. Polypeptides, including the provided receptors and otherpolypeptides, e.g., linkers or peptides, may include amino acid residuesincluding natural and/or non-natural amino acid residues. The terms alsoinclude post-expression modifications of the polypeptide, for example,glycosylation, sialylation, acetylation, and phosphorylation. In someaspects, the polypeptides may contain modifications with respect to anative or natural sequence, as long as the protein maintains the desiredactivity. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the proteins or errors due to PCRamplification.

As used herein, a “subject” is a mammal, such as a human or otheranimal, and typically is human. In some embodiments, the subject, e.g.,patient, to whom the agent or agents, cells, cell populations, orcompositions are administered, is a mammal, typically a primate, such asa human. In some embodiments, the primate is a monkey or an ape. Thesubject can be male or female and can be any suitable age, includinginfant, juvenile, adolescent, adult, and geriatric subjects. In someembodiments, the subject is a non-primate mammal, such as a rodent.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to complete or partial amelioration orreduction of a disease or condition or disorder, or a symptom, adverseeffect or outcome, or phenotype associated therewith. Desirable effectsof treatment include, but are not limited to, preventing occurrence orrecurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis.The terms do not imply complete curing of a disease or completeelimination of any symptom or effect(s) on all symptoms or outcomes.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. In some embodiments, sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe disease. For example, a late stage cancer, such as development ofmetastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. In some embodiments, the provided cells and compositionsare used to delay development of a disease or to slow the progression ofa disease.

As used herein, to “suppress” a function or activity is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, cells that suppress tumor growthreduce the rate of growth of the tumor compared to the rate of growth ofthe tumor in the absence of the cells.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,cells, or composition, in the context of administration, refers to anamount effective, at dosages/amounts and for periods of time necessary,to achieve a desired result, such as a therapeutic or prophylacticresult.

A “therapeutically effective amount” of an agent, e.g., a pharmaceuticalformulation or cells, refers to an amount effective, at dosages and forperiods of time necessary, to achieve a desired therapeutic result, suchas for treatment of a disease, condition, or disorder, and/orpharmacokinetic or pharmacodynamic effect of the treatment. Thetherapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the subject, and thepopulations of cells administered. In some embodiments, the providedmethods involve administering the cells and/or compositions at effectiveamounts, e.g., therapeutically effective amounts.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount. In the context of lower tumor burden, theprophylactically effective amount in some aspects will be higher thanthe therapeutically effective amount.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.”

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

As used herein, a composition refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, “enriching” when referring to one or more particularcell type or cell population, refers to increasing the number orpercentage of the cell type or population, e.g., compared to the totalnumber of cells in or volume of the composition, or relative to othercell types, such as by positive selection based on markers expressed bythe population or cell, or by negative selection based on a marker notpresent on the cell population or cell to be depleted. The term does notrequire complete removal of other cells, cell type, or populations fromthe composition and does not require that the cells so enriched bepresent at or even near 100% in the enriched composition.

As used herein, a statement that a cell or population of cells is“positive” for a particular marker refers to the detectable presence onor in the cell of a particular marker, typically a surface marker. Whenreferring to a surface marker, the term refers to the presence ofsurface expression as detected by flow cytometry, for example, bystaining with an antibody that specifically binds to the marker anddetecting said antibody, wherein the staining is detectable by flowcytometry at a level substantially above the staining detected carryingout the same procedure with an isotype-matched control or fluorescenceminus one (FMO) gating control under otherwise identical conditionsand/or at a level substantially similar to that for cell known to bepositive for the marker, and/or at a level substantially higher thanthat for a cell known to be negative for the marker.

As used herein, a statement that a cell or population of cells is“negative” for a particular marker refers to the absence of substantialdetectable presence on or in the cell of a particular marker, typicallya surface marker. When referring to a surface marker, the term refers tothe absence of surface expression as detected by flow cytometry, forexample, by staining with an antibody that specifically binds to themarker and detecting said antibody, wherein the staining is not detectedby flow cytometry at a level substantially above the staining detectedcarrying out the same procedure with an isotype-matched control orfluorescence minus one (FMO) gating control under otherwise identicalconditions, and/or at a level substantially lower than that for cellknown to be positive for the marker, and/or at a level substantiallysimilar as compared to that for a cell known to be negative for themarker.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

Unless defined otherwise, all terms of art, notations and othertechnical and scientific terms or terminology used herein are intendedto have the same meaning as is commonly understood to which the claimedsubject matter pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood.

All publications, including patent documents, scientific articles anddatabases, referred to in this application are incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication were individually incorporated by reference. If adefinition set forth herein is contrary to or otherwise inconsistentwith a definition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth herein prevails over the definitionthat is incorporated herein by reference.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

IX. Examples

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1: Administration of Anti-CD19 CAR-Expressing Cells to Subjectswith Relapsed and Refractory Non-Hodgkin's Lymphoma (NHL)

A. Subjects and Treatment

Therapeutic CAR⁺ T cell compositions containing autologous T cellsexpressing a chimeric antigen-receptor (CAR) specific for CD19 wereadministered to subjects with B cell malignancies.

Example 1.A.1

Results are described in this Example 1.A.1 for evaluation through aparticular time-point (1.A.1) in an ongoing clinical study administeringsuch therapy to patients with B cell Malignancies. Specifically, acohort (full cohort) of (at this time-point, fifty-five (55)) adulthuman subjects with relapsed or refractory (R/R) aggressivenon-Hodgkin's lymphoma (NHL), including diffuse large B-cell lymphoma(DLBCL), de novo or transformed from indolent lymphoma (NOS), high-gradeB-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements with DLBCLhistology (double/triple hit), DLBCL transformed from chroniclymphocytic leukemia (CLL) or marginal zone lymphomas (MZL), primarymediastinal large b-cell lymphoma (PMBCL), and follicular lymphoma grade3b (FL3B) after failure of 2 lines of therapy. Among the subjectstreated were those having Eastern Cooperative Oncology Group (ECOG)scores of between 0 and 2 (median follow-up 3.2 months). The full cohortdid not include subjects with mantle cell lymphoma (MCL). No subjectswere excluded based on prior allogeneic stem cell transplantation (SCT),secondary central nervous system (CNS) involvement or an ECOG score of2, and there was no minimum absolute lymphocyte count (ALC) forapheresis required.

Outcomes were separately assessed for a core subset of subjects withinthe full cohort (subjects within the full cohort excluding thosesubjects with a poor performance status (ECOG 2), DLBCL transformed frommarginal zone lymphomas (MZL) and/or chronic lymphocytic leukemia (CLL,Richter's), and subjects with primary mediastinal large b-cell lymphoma(PMBCL), and follicular lymphoma grade 3b (FL3B) (core cohort)). Thecore cohort includes subjects with DLBCL, NOS and transformed follicularlymphoma (tFL) or high grade B-cell lymphoma (double/triple hit) orhigh-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangementswith DLBCL histology (double/triple hit) and with Eastern CooperativeOncology Group performance status (ECOG PS) of 0 or 1 At the timepointin Example 1.A.1, outcomes for 44 subjects within this core cohort wereassessed.

The demographics and baseline characteristics of the full and corecohort subjects at the timepoint in Example 1.A.1 are set forth in TableE1.

TABLE E1 Demographics and Baseline Characteristics FULL CORECharacteristic N = 55 N = 44 Median Age, years (range) 61 (29-82) 61(29-82) ≥65 years, n (%) 22 (40) 17 (39) Male/Female, n (%) 38/17(69/31) 28/16 (64/36) Months from diagnosis, median (range) 17 (3-259)20 (8-259) B-NHL Subtype, n (%) DLBCL, NOS 40 (73) 35 (80) TransformedDLBCL 14 (26)  8 (18) Follicular, Grade 3B 1 (2) 1 (2) MolecularSubtype, n (%) Double/triple hit 15 (27) 12 (27) Double expressor  6(11) 4 (9) Patient Characteristics, n (%) Chemorefractory^(†) 42 (76) 34(77) ECOG 0-1 48 (87)  44 (100) ECOG 2  7 (13) 0 Prior lines of therapy,median (range) 3 (1-11) 3 (1-8) <5 lines of therapy 44 (80) 37 (84) AnyHSCT 27 (49) 22 (50) Allogeneic 4 (7) 3 (7) Autologous 24 (44) 20 (45)*SD or PD to last chemo-containing regimen or relapse <12 months afterautologous SCT

The therapeutic T cell compositions administered had been generated by aprocess including immunoaffinity-based (e.g., immunomagnetic selection)enrichment of CD4⁺ and CD8⁺ cells from leukapheresis samples from theindividual subjects to be treated. Isolated CD4⁺ and CD8⁺ T cells wereseparately activated and independently transduced with a viral vector(e.g., lentiviral vector) encoding an anti-CD19 CAR, followed byseparate expansion and cryopreservation of the engineered cellpopulations in a low-volume. The CAR contained an anti-CD19 scFv derivedfrom a murine antibody (variable region derived from FMC63,V_(L)-linker-V_(H) orientation), an immunoglobulin-derived spacer, atransmembrane domain derived from CD28, a costimulatory region derivedfrom 4-1BB, and a CD3-zeta intracellular signaling domain. The viralvector further contained sequences encoding a truncated receptor, whichserved as a surrogate marker for CAR expression; separated from the CARsequence by a T2A ribosome skip sequence.

The cryopreserved cell compositions were thawed prior to intravenousadministration. The therapeutic T cell dose was administered as adefined cell composition by administering a formulated CD4⁺ CAR⁺ cellpopulation and a formulated CD8⁺ CAR⁺ population administered at atarget ratio of approximately 1:1. Subjects were administered a singleor double dose of CAR-expressing T cells (each single dose via separateinfusions of CD4⁺ CAR-expressing T cells and CD8⁺ CAR-expressing Tcells, respectively) as follows: a single dose of dose level 1 (DL-1)containing 5×10⁷ total CAR-expressing T cells (n=30 for subjectsassessed in Example 1.A.1), a double dose of DL1 in which each dose wasadministered approximately fourteen (14) days apart (n=6 for subjectsassessed in Example 1.A.1, administered on day 1 and day 14, includingone subject that inadvertently received two DL2 doses via the two-doseschedule, due to a dosing error), or a single dose of dose level 2(DL-2) containing 1×10⁸ total CAR-expressing T cells (n=18 for subjectsassessed in Example 1.A.1). The target dose level and the numbers of Tcell subsets for the administered compositions are set forth in TableE2.

TABLE E2 Target dose levels and number of T cell subsets for cellcompositions containing anti-CD19 CAR T cells Dose Helper T cell (T_(H))Cytotoxic T Cell (T_(C)) Total T Cell Dose level Dose (CD4⁺CAR⁺) Dose(CD8⁺CAR⁺) (CD3⁺ CAR⁺) 1 25 × 10⁶ 25 × 10⁶  50 × 10⁶ 2 50 × 10⁶ 50 × 10⁶100 × 10⁶

Beginning at prior to CAR⁺ T cell infusion, subjects received alymphodepleting chemotherapy with fludarabine (flu, 30 mg/m²) andcyclophosphamide (Cy, 300 mg/m²) for three (3) days. The subjectsreceived CAR-expressing T cells 2-7 days after lymphodepletion.

Example 1.A.2

For Example 1.A.2, at a subsequent point in time in the clinical studydescribed in this Example 1 above, results were analyzed. At thisanalysis time point in Example 1.A.2, 74 patients had been treated (51male, 23 female). The subjects included sixty-nine (69) subjects in thefull DLBCL cohort (including 67 DLBCL NOS (45 de novo, 14 transformedfrom FL, 8 transformed from CLL or MZL), 1 FL grade 3B, and 1 PMBCL),and 5 subjects in the MCL cohort. Among subjects in the full (DLBCL)cohort, median age was 61 yrs (range 26, 82), median prior therapies was3 (range 1, 12), 46 (67%) were chemorefractory, 32 (46%) had any priortransplant, and at least 16 (23%) patients had double/triple hitlymphoma. Forty-nine (49) subjects in the core cohort were assessed atthis timepoint in 1.A.2.

B. Safety

The presence or absence of treatment-emergent adverse events (TEAE)following administration of the CAR-T cell therapy was assessed.Subjects also were assessed and monitored for neurotoxicity(neurological complications including symptoms of confusion, aphasia,encephalopathy, myoclonus seizures, convulsions, lethargy, and/oraltered mental status), graded on a 1-5 scale, according to the NationalCancer Institute—Common Toxicity Criteria (CTCAE) scale, version 4.03(NCI-CTCAE v4.03). Common Toxicity Criteria (CTCAE) scale, version 4.03(NCI-CTCAE v4.03). See Common Terminology for Adverse Events (CTCAE)Version 4, U.S. Department of Health and Human Services, Published: May28, 2009 (v4.03: Jun. 14, 2010); and Guido Cavaletti & Paola MarmiroliNature Reviews Neurology 6, 657-666 (December 2010). Cytokine releasesyndrome (CRS) also was determined and monitored, graded based onseverity. See Lee et al, Blood. 2014; 124(2):188-95. In some cases,adverse events data were reported and collected starting atlymphodepletion to 90 days after CAR⁺ T cell administration.

Example 1.B.1

Example 1.B.1 describes results based on the analysis time-point inExample 1.A.1.

FIG. 1 depicts the percentage of such subjects who were observed to haveexperienced laboratory abnormalities and TEAEs, which occurred in ≥20%of subjects. In addition to the TEAEs shown in FIG. 1 , the followingevent terms were observed at Grade 3-4 in ≥5% of patients: white bloodcell count decreased (13.6%), encephalopathy (12%), hypertension (7%).Degree of toxicities observed were consistent between dose levels 1 and2.

In 84% of the full cohort subjects in Example 1.B.1 analysis, severe(grade 3 or higher) cytokine release syndrome (CRS) and severeneurotoxicity were not observed. Additionally, it was observed that 60%of the full cohort subjects did not develop any grade of CRS orneurotoxicity. No differences in incidence of CRS, neurotoxicity (NT),sCRS, or severe neurotoxicity (sNT) were observed between dose levels.Table E3 summarizes the incidence of cytokine release syndrome (CRS) andneurotoxicity adverse events in patients 28 days after receiving atleast one dose of CAR-T cells. As shown in Table E3, no sCRS (Grade 3-4)was observed in any subjects that received a single dose of DL2 ordouble dose of DL1. Severe neurotoxicity or severe CRS (grade 3-4) wasobserved in 16% (9/55) of the full cohort of subjects and in 18% (8/44)of the subjects in the core subset. 11% (n=6) of subjects receivedtocilizumab, 24% (n=13) of subjects received dexamethasone. Among theECOG2 subjects within the full cohort, observed rates of CRS andneurotoxicity were 71% and 29%, respectively.

TABLE E3 Assessment of Presence or Absence of CRS and NeurotoxicityAdverse Events for Example 1.B.1 FULL All Dose Levels DL1S DL2S DL1D^(†)CORE Safety, N 55 30 19 6 44 sCRS or sNT, n (%)  9 (16)  6 (20) 2 (11) 1(17)  8 (18) CRS or NT, n (%) 22 (40) 12 (40) 7 (37) 3 (50) 15 (34) CRSGrade 1-2, n (%) 18 (33) 10 (33) 5 (26) 3 (50) 12 (27) Grade 3-4, n (%) 1 (2) 1 (3) 0 0 1 (2) Neurotoxicity Grade 1-2, n (%) 3 (6) 1 (3) 2 (11)0 2 (5) Grade 3-4, n (%)  9 (16)  6 (20) 2 (11) 1 (17)  8 (18)^(†)Includes one patient treated at DL2 2-dose schedule due to dosingerror

FIG. 2 shows a Kaplan meier curve depicting observed time to onset ofCRS and/or neurotoxicity for the analysis in 1.B.1. As shown, theobserved median times to onset of CRS and to onset of neurotoxicity were5 and 11 days, respectively, with only 11% of patients experiencingonset of CRS less than 72 hours after initiation of the administrationof the cell therapy. The median time to resolution of CRS andneurotoxicity to Grade 1 or better was 5 and 7 days, respectively. Themedian time to complete resolution of CRS and neurotoxicity was 5 and 11days, respectively. The results were consistent with a conclusion thatthere was a low rate of early onset of any CRS or neurotoxicity in thesubjects.

Example 1.B.2

Example 1.B.2 describes assessment at the time-point in Example 1.B.2.Up to this time point, adverse event (AE) data were collected fromlymphodepletion (LD) to 90 days post administration of CAR-expressing Tcells. At the second time point, 69 subjects in the DLBCL cohort (fullcohort) were evaluated for safety, 38 that had received DL1 single dose,25 having received DL2 single dose, and 6 having received DL1 doubledose schedule. The most common TEAEs other than CRS or NT includedneutropenia (41%, 28/69), fatigue (30%, 21/69), thrombocytopenia (30%,21/69), and anemia (26%, 18/69). One Grade 5 TEAE of diffuse alveolardamage was observed.

No acute infusional toxicity was observed, and the majority of subjectsin the full cohort, 64% (44/69), were observed to have no CRS or NT,indicating that outpatient delivery of CAR-expressing T cells may bepossible. Rates of CAR T cell-associated toxicities, including CRS andNT, did not differ between dose levels. Safety profile was observed tobe similar across cohorts and dose levels. Among the 25 subjects in thefull cohort (36%) who experienced any grade CRS or NT, 21 (30%) had CRSand 14 (20%) had NT. No subjects had Grade 3 CRS and only one (1%, 1/69)had Grade 4 CRS and required ICU care; the other 29% (20/69) had Grade1-2 CRS. Of the 20% of subjects with NT, 6% (4/69) had Grade 1-2 and 14%(10/69) had Grade 3-4; 2 (3%) had seizure. No Grade 5 CRS or grade 5 NTwas observed. No incidences of cerebral edema were observed. All CRS andNT events were resolved except one case of Grade 1 tremor, which wasongoing at the time of analysis. Median time to onset of first CRS andNT was 5 days (range 2, 12) and 10 days (range 5, 23), respectively. Inthe first 72 hours post infusion, no subjects were observed to have NT,and only 10% (7/69) were observed to have CRS (all Grade 1); NT waspreceded by CRS in >70% of subjects. Overall, thirteen (13) subjects(19%) required intervention for CRS or NT with anti-cytokine therapy(tocilizumab alone 1 (1%), dexamethasone alone 6 (9%), or both 6 (9%))and only one required any vasopressor support. Median doses oftocilizumab and dexamethasone were 1 and 6, respectively. Median CRS andNT duration was 5 days and 11 days, respectively. Analysis of the corecohort (n=49) also showed similar rates of CRS and NT.

In this assessment, low incidences and late onsets of CRS and/or NT wereobserved, at both dose levels, supported the feasibility of outpatientinfusion, such as with hospital admission at the first sign of fever orfever lasting beyond a certain period of time. No Grade 5 CRS or grade 5NT was observed, and all severe CRS and severe NT were resolved.Further, approximately 2 out of 3 patients had no CRS or NT, supportingthat the cells can be administered on outpatient basis. At the time ofassessment in 1.B.2, four subjects had been treated in the outpatientsetting. Further, no meaningful differences in toxicity was observed insubjects receiving DL1 or DL2, indicating achievement of higher responserates without an increase risk of toxicity or safety concerns.

C. Response Outcomes Following Treatment

Subjects were monitored for response, including by assessing tumorburden at 1, 3, 6, 7, 12, 18, and 24 months after administration of theCAR⁺ T cells.

Example 1.C.1

Example 1.C.1 describes results based on the analysis time-point inExample 1.A.1 and 1.B.1.

Response rates are listed in Table E4. High durable response rates wereobserved in the cohort of subjects, which included subjects heavilypretreated or, with poor prognosis and/or with relapsed or refractorydisease. For subjects across all doses in the Core (n=44) cohort, theobserved overall response rate (ORR) was 86% and the observed completeresponse (CR) rate was 59%. At three months for the core cohort, theoverall response rate (ORR) was 66%; the three-month CR rate was 50%among the core cohort. In the core cohort, the 3 month ORR was 58%(11/19) at dose level 1 and 78% at dose level 2; the 3 month CR rate was42% (8/19) for dose level 1 and 56% (5/9) for dose level 2, consistentwith a suggested dose response effect on treatment outcome.Additionally, the results were consistent with a relationship betweendose and durability of response.

TABLE E4 Response FULL CORE All Dose All Dose Levels DL1S DL2S DL1D^(c)Levels DL1S DL2S DL1D^(a) Best Overall 54 30 18 6 44 25 15 4 Response,N^(a) ORR, % 76 (62, 87) 80 (61, 92) 72 (47, 90) 67 (23, 96) 86 (73, 95)84 (64, 95) 87 (60, 98) 100 (40, 100) (95% CI) CR, % 52 (38, 66) 53 (34,72) 50 (26, 74) 50 (12, 88) 59 (43, 74) 56 (35, 76) 60 (32, 84) 75 (19,99) (95% CI) ≥3 mos f/u, n^(b) 41 24 11 6 32 19  9 4 3 mo ORR, % 51 (35,67) 46 (26, 67) 64 (31, 89) 50 (12, 88) 66 (47, 81) 58 (34, 80) 78 (40,97) 75 (19, 99) (95% CI) 3 mo CR, % 39 (24, 56) 33 (16, 55) 46 (17, 77)50 (12, 88) 50 (32, 68) 42 (20, 67) 56 (21, 86) 75 (19, 99) (95% CI)DL1S: DL1 1-dose schedule; DL2S: DL2 1-dose schedule; DL1D: DL1 2-doseschedule; ^(a)Included patients with event of PD, death, or 28 dayrestaging scans. Treated patients 28 days prior to data snapshot werenot included. ^(b)The denominator is number of patients who received theCAR T-cell therapy ≥3 months ago, prior to snapshot date with anefficacy assessment at Month 3 or prior assessment of PD or death.^(c)Includes one patient treated at DL2 2-dose schedule due to dosingerror

Overall response rates among various subgroups of subjects in the fulland core cohorts are shown in FIGS. 3A and 3B, respectively. Inpoor-risk DLBCL subgroups, response rates were generally high. An ORR ofgreater than 50% was observed at 3 months in patients with double/triplehit molecular subtype, that had primary refractory or chemorefractoryDLBCL or that never before had achieved a CR. Complete resolution of CNSinvolvement by lymphoma was observed in 2 patients.

Among the subjects treated six months or greater prior to the particulartime-point of the evaluation, of the ten (10) patients that had been inresponse at three months, 9 (90%) remained in response at six months. Atthe evaluation time-point, 97% of subjects in the core subset who hadresponded were alive and in follow-up, median follow-up time 3.2 months.

Results for the duration of response and overall survival (grouped bybest overall response (non-responder, CR/PR, CR and/or PR)) are shownfor full and core cohorts of subjects, in FIGS. 4A and 4B, respectively.As shown, prolonged survival was observed in responders, with increaseddurability of response in subjects with CRs. All patients in response atthree months remained alive at the time of evaluation, although 5/6subjects with poor performance status (ECOG 2) had expired.

FIG. 8 shows a graph plotting progression-free time (months) forindividual subjects within the full and core cohorts. Each barrepresents a single patient. Shading indicates best overall response (ineach case, unless otherwise indicated, achieved at 1 month); textureindicates dose (solid=dose level 1, single dose; cross-hatched,dose-level 2, single dose; vertical hatched=dose level 1, two-dose).Horizontal arrows indicate an ongoing response. Certain individualsubjects were initially assessed (e.g., at 1-month) as exhibiting stabledisease (SD) or Partial Response (PR), and were later observed to haveachieved a PR (e.g., conversion of SD to PR) or CR. In such cases,shading of the individual patient bar, as noted, indicates best overallresponse, and dots (same correspondence of shading to response achieved)along each individual subject bar, indicate when each SD, PR, and/or CRwas observed to have occurred in the subject.

Complete resolution of CNS involvement by lymphoma was observed in twopatients. CAR⁺ cells in one subject were observed to have expandedfollowing biopsy after relapse. The subject who exhibited expansionfollowing biopsy had chemorefractory transformed DLBCL (germinal centersubtype with a BCL2 rearrangement and multiple copies of MYC and BCL6).The subject had been administered the CAR⁺ T cells at DL-1 and thenumbers of CD3⁺/CAR⁺, CD4⁺/CAR⁺, CD8⁺/CAR⁺ T cells in peripheral bloodwere measured at certain time points, are shown in FIG. 6A. The subjecthad previously been treated with, and was refractory to, five priorlines of therapy including dose-adjusted etoposide, doxorubicin, andcyclophosphamide with vincristine and prednisone plus rituximab(DA-EPOCH-R) and intermediate-intensity allogeneic stem-celltransplantation from an 8/8 HLA-matched unrelated donor. Followingallogeneic stem cell transplantation and prior to receiving CAR⁺ Tcells, the subject showed 100% donor chimerism in all blood lineages,had ceased taking immunosuppressive therapy, and did not have graftversus host disease (GVHD). Prior to administration of CAR⁺ T cells, thesubject had a periarticular mass and temporal lobe lesion observed bypositron-emission tomography and computed tomography (PET-CT) (FIG. 6B)and confirmed by magnetic resonance imaging (MRI) (FIG. 6D).

After receiving anti-CD19 CAR-T cell treatment, the subject achieved CR28 days post-infusion, as shown by PET-CT (FIG. 6C) and brain MRI (FIG.6E), with no observed signs of neurotoxicity or CRS. Three monthspost-infusion of the CAR-T cells, relapse of the periarticular mass wasnoted in this subject (FIG. 6F), and an incisional biopsy was performed.As shown in FIG. 6A, following biopsy, the visible tumor receded with nofurther therapy. Pharmacokinetic analysis showed a marked re-expansionof the CAR⁺ T cells in peripheral blood (to a level higher than initialexpansion observed, with peak levels observed at about 113 dayspost-infusion) i, which coincided with tumor regression. The subjectthen went on to achieve a second CR, as confirmed by restaging PET-CTone month following the biopsy (FIG. 6G), and remained in CR at 6 monthspost CAR-T cell infusion. Further assessment of the subject showed thatthe CNS response was durable and the subject remained in CR at 12months.

The results in the subject having received the biopsy followed byobserved re-expansion of CAR⁺ T cells are consistent with a conclusionthat re-expansion and activation of CART cells can be initiated in vivofollowing reduction or loss of functional or active CAR⁺ T cells and/orrelapse following anti-tumor response to CAR-T cell therapy. Further,following re-expansion in vivo late after initial CAR⁺ T cell infusion,the CAR⁺ T cells are able to re-exert anti-tumor activity. This resultsupports that CAR⁺ T cell re-expansion and activation can be triggeredin vivo and that methods of reactivating CAR⁺ T cells, may furtheraugment their efficacy.

The complete responses in the two DLBCL subjects with CNS involvementwere observed without development of any grade of neurotoxicity. Theseresults are consistent with the observation that CAR⁺ T cells ofembodiments provided herein are capable of readily accessing the CNS andexerting effector function to reduce or eliminate CNS tumors, withoutincreasing or without substantially increasing risk of toxicity such asneurotoxicity. In other studies, among subjects having ALL treated withanti-CD19 CAR T cells, no clear correlation has been observed betweenincidence of neurotoxicity and the presence of CNS leukemia in the brain(which has been observed to respond to such CAR T cell therapy). Thus,whereas neurotoxicity can occur in some contexts following treatmentwith CAR-T therapies, such neurotoxicity may not necessarily be theresult of target expression in the brain or activity of the CAR T cellsin the CNS, and may not result from “on-target” toxicity by the CAR⁺ Tcells.

Example 1.C.2

Example 1.C.2 describes results based on the analysis time-point inExample 1.A.2 and 1.B.2.

Up to the time point in Example 1.C.2, 68 subjects in the full DLBCLcohort was evaluated for response. Overall or objective response (OR),3-month, and 6-month objective response rates were 75% (51/68), 49%(27/55), and 40% (14/35), respectively. Complete response (CR) rate,3-month CR rate, and 6-month CR rate were 56% (38/68), 40% (22/55), and37% (13/35), respectively. A trend toward improved response rate at 3months was observed in subjects treated at DL2 compared to DL1: 63%(12/19; 95% CI 38, 84) vs 40% (12/30; 95% CI 23, 59) for ORR withp=0.148, and 58% (11/19; 95% CI 34, 80) vs 27% (8/30; 95% CI: 12, 46)for CR with p=0.0385. Among 16 double/triple hit lymphoma subjects, ORRwas 81%, and 3-month CR rate was 60%.

In the core cohort (n=49 for the time-point in Example 1.C.2), OR,3-month, and 6-month OR rates were 84% (41/49), 65% (26/40), and 57%(13/23), respectively. CR rate, 3-month CR rate, and 6-month CR ratewere 61% (30/49), 53% (21/40), and 52% (12/23), respectively. A similartrend in improved durable ORR and CR at 3 months at higher doses wasobserved. Specifically, for patients in the CORE cohort administeredDL2, 3-month ORR was 80% (12/15; 95% CI 52, 96) and 3-month CR was 73%(11/15; 95% CI 45, 92), compared to 3-month ORR and CR rates of 52%(11/21; 95% CI 30, 74) and 33% (7/21; 95% CI 15, 57) in CORE cohortsubjects administered DL1, with p=0.159 and p=0.0409 respectively. Amongsubjects in the CORE cohort having received DL2 and with 3-monthfollow-up (n=15), 3-month ORR was 80% and 3-month CR was 73%.

Median DOR in the full cohort and core cohorts at this time-point in1.C.2 was 5.0 and 9.2 months, respectively; median duration of CR was9.2 months in the full cohort. Median duration of CR had not beenreached in the core cohort. Median overall survival (OS) was 13.7 monthsin the full cohort and had not been reached in the core cohort. 6-monthOS was 75% in the full cohort, with median follow-up of 5.8 months.6-month OS was 88% in the core cohort, with median follow up of 5.6months.

D. Assessment of CAR⁺ T Cells in Blood

Based on data from the time-point described in Example 1.A.1, 1.B.1 and1.C.1, pharmacokinetic analysis was carried out to assess numbers ofCAR⁺ T cells in peripheral blood at various time points post-treatment.Results from the fifty-five (55) subjects assessed at the time-point inExample 1.A.1 in the DLBCL cohort and four (4) subjects (assessed atthat same time-point) in the mantle cell lymphoma (MCL) cohort, asdescribed in Example 2 below. were analyzed. Pharmacokinetics (PK)measurements were carried out using validated flow cytometry to detect amarker expressed in the CAR construct and quantitative PCR-based assaysto detect the integration of the CAR construct. B cell aplasia wasassessed by flow cytometry using anti-CD19 antibodies. As shown in FIG.5A, CD4⁺ and CD8⁺ CAR-expressing cells, as measured by the number ofcells/μL blood (median ±quartiles) plotted on a log scale, were detectedthroughout the course of assessment at both administered dose levels.Subjects receiving DL2 relative to DL1 had higher median C_(max) andmedian AUC_(0.28) for CD3⁺/CAR⁺, CD4⁺/CAR⁺, and CD8⁺/CAR⁺ T cell subsetsin peripheral blood (AUC₀₋₂₈: DL2 vs. DL1 was 1836 vs. 461, 350 vs. 182,and 1628 vs. 114, for CD3⁺, CD4⁺, and CD8⁺, respectively; p<0.05 forCD8⁺; C_(max): DL2 vs. DL1 was 99.8 vs. 27.9, 15.1 vs. 5.2, and 73.1 vs.5.5 cells/μL, respectively). Median time to maximum CD3⁺ CAR⁺ T cellexpansion was 15 days (range 8-29) and did not differ between doselevels. CD4⁺ and CD8⁺ CAR-expressing T cells homed to the bone marrow atrelatively similar levels.

An increased median area under the curve (AUC) (CD8⁺ CAR⁺ T cell numbersover time in the blood) was observed among subjects administered thehigher dose level, as compared to the lower dose level, without anobserved increase in toxicity. Higher peak CD8⁺/CAR⁺ T cell exposure wasobserved in responders (CR/PR) than non-responders (PD); persistence ofcells over the time of assessment, including out to 3 and 6 months, wasobserved even in subjects whose disease had progressed (FIG. 5B). MedianC_(max) and median AUC₀₋₂₈ of CD8⁺ CAR⁺ T cells were higher inresponding subjects and with durable response at month 3 (CD8⁺ C_(max)median=20.8 vs. 5.5; CD8⁺ AUC₀₋₂₈ median=235 vs. 55 in CR/PR at Month 3vs. PD at Month 3). Among subjects that were evaluated for CAR T cellpersistence, 90% and 93% of 29 subjects had detectable CD8⁺ and CD4⁺CAR⁺ T cells, respectively, at month 3; 63% and 58% of 19 subjects haddetectable CD8⁺ and CD4⁺ CAR⁺ T cells, respectively, at month 6. Atmonths 3 and 6, no statistically significant differences in thepersistence of CAR⁺ T cells were observed between subjects with durableresponse or relapse. CAR⁺ T cells were detectable at time of relapse in89% of 11 subjects with PK, even though B cell aplasia (<1 cell/p1) wasdemonstrated in nearly all subjects 97% (34/35) at month 3, and 100%(24/24) at month 6.

Higher C_(max) and AUC₀₋₂₈ at DL2 as compared to DL1 was not observed tobe associated with increased CRS or NT. For any NT or for >Grade 2 CRS,median AUCs of CD4⁺/CAR⁺ and CD8⁺/CAR⁺ T cells were 5 to 10 fold and 3to 5 fold higher, respectively, than the median AUC for DL2. Higherdisease burden and baseline levels of inflammatory cytokines wasobserved to be associated with higher peak levels of CAR⁺ T cells,higher cytokine peak levels, and higher incidences of CRS and NT. Theresults were consistent with a conclusion that the higher C_(max) andmedian AUC₀₋₂₈ at DL2 did not increase CRS or NT.

The results were consistent with a conclusion that treatment resulted inprolonged exposure and persistence of the engineered cells, even insubjects with poor responses. In some embodiments, combinationapproaches are used, such as administration of an immune checkpointmodulator or other immune modulatory agent, e.g., following relapse ordisease progression, at a time at which engineered cells persist in thesubject, e.g., as measured by levels of cells in peripheral blood. Insome aspects, the cells, having persisted for a prolonged period,re-expand or become activated and/or exhibit anti-tumor function,following administration of the other agent or treatment. Higher medianCD4⁺ and CD8⁺ CAR⁺ T cell numbers were generally observed over time inblood of subjects who developed neurotoxicity (FIG. 5C). Resultsindicated that the CAR⁺ T cells exhibited expansion and persistence,durability of response at 3 months that increased at higher dose levels,without increased toxicity. Results were observed that were consistentwith a suggestion that high peak levels of CAR⁺ T cells and cytokines inthe blood may be associated with NT and CRS, and may be influenced bybaseline subject factors. It was observed that CAR⁺ T cells were presentat the time of relapse, indicating that combination or retreatmentapproaches may provide certain advantages.

E. Blood Analytes and Neurotoxicity, CRS and Response

Various pre-treatment blood analytes, including cytokines, were measuredin the serum of subjects (those assessed at the time-point in Example1.A.1), prior to administration of the CAR⁺ T cells. Cytokines weremeasured using a multiplex cytokine assay. Potential correlations torisk of developing neurotoxicity were assessed using statisticalanalysis based on univariate nonparametric tests.

FIG. 7 shows median levels of the assessed analytes in units (LDH, U/L;ferritin, ng/mL; CRP, mg/L; cytokines, pg/mL) in subjects that did notdevelop a neurotoxicity versus subjects that did develop a neurotoxcityfollowing CAR⁺ T cell therapy. Levels of certain blood analytes,including LDH, Ferritin, CRP, IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1and MIP-1β, were observed to be associated with level of risk ofdeveloping neurotoxicity (Wilcoxon p values <0.05,without multiplicityadjustment). In particular, the results were consistent with aconclusion that pre-treatment levels of LDH, which in some embodimentsis a surrogate for disease burden, may be useful for potentialneurotoxicity risk assessment and/or risk-adapted dosing or adjustmentof treatment of certain subjects. In addition, tumor burden measuredbefore administration of the CAR-T cell composition correlated (Spearmanp values <0.05) with the risk of developing neurotoxicity. In someaspects, LDH levels may be assessed alone and/or in combination withanother pre-treatment parameter, such as another measure or indicator ofdisease burden, such as a volumetric tumor measurement such as sum ofproduct dimensions (SPD) or other CT-based or MRI-based volumetricmeasurement of disease burden. In some aspects, one or more parametersindicative of disease burden are assessed, and in some contexts mayindicate the presence, absence or degree of risk of developingneurotoxicity following the T cell therapy. In some aspects, the one ormore parameters include LDH and/or a volumetric tumor measurement.

In an additional analysis, fifty-five (55) subjects in the DLBCL cohortat the time-point in Example 1.A.1, and four (4) subjects in the mantlecell lymphoma (MCL) described in Example 2 below were included inanalysis for correlation with safety evaluations. In the 59 subjectsevaluated for safety, CRS was observed in 32% (30% Grade 1-2, 0% Grade3, 2% Grade 4); NT was observed in 20% (5% Grade 1-2, 10% Grade 3, 5%Grade 4). Dose level did not correlate with CRS or NT (p=0.565 andp=1.00, respectively). Subject factors that correlate with any grade CRSand NT were poorer performance status (e.g. ECOG Status 2) (p=0.03) andhigher disease burden (p<0.05) as measured by the sum of the products ofdiameters (SPD) based on imaging results. Pre-CAR⁺ T cell infusionclinical laboratory parameters and cytokine measurements for pre-CAR⁺ Tcell infusion that were observed to be associated with the occurrence ofany grade NT included higher serum LDH, ferritin, and CRP, and higherplasma IL-6, IL-8, IL-10, TNF-α, IFN-α2, MCP-1, and MIP-1θ (p<0.05 foreach). Higher pre-CAR⁺ T cell infusion plasma levels of IL-8, IL-10, andCXCL10 were also associated with Grade 3-4 NT (p<0.05 for each).

Of the 54 subjects in the DLBCL cohort that were evaluated for response,higher ECOG scores and DLBCL transformed from CLL or MZL correlated withlower durable response at month 3 (p=0.02 for both). Pre-CAR⁺ T cellinfusion parameters associated with best ORR included lower values offerritin, LDH, CXCL10, G-CSF, and IL-10, and those associated withdurable response at 3 months included lower ferritin, CRP, LDH, CXCL10,IL-8, IL-10, IL-15, MCP-1β, TNF-α, and higher pre-CAR⁺ T infusionhemoglobin and albumin (p<0.05 for each).

In some cases, the apheresis sample and CAR⁺ T cell composition foradministration was assessed and correlated with clinical outcomes. Theresults showed that T cell memory subsets and T cell functionality maycorrelate with certain clinical outcomes.

The results showed that certain baseline patient characteristics,including inflammatory state and high tumor burden prior to treatment,may be useful for the identification of patients at risk for increasedtoxicity following administration of CAR-expressing T cells. Low tumorburden and low inflammatory state were observed to be associated withimproved toxicity profile and better durability of response. The resultssupport that treating subjects earlier in the course of therapy and/orassessing a panel of clinical and laboratory biomarkers to risk stratifysubjects for potential early intervention may mitigate the risk oftoxicity and improve durability of response.

Example 2: Administration of Anti-CD19 CAR-Expressing Cells to Subjectswith Mantle Cell Lymphoma (MCL)

Therapeutic CAR⁺ T cell compositions containing autologous T cellsexpressing a chimeric antigen-receptor (CAR) specific for CD19,generated as described in Example 1, were administered to four (4) humansubjects with mantle cell lymphoma (MCL) that had failed 1 line oftherapy. The cryopreserved cell compositions were thawed prior tointravenous administration. The therapeutic T cell composition wasadministered as a defined composition cell product with formulated CD4⁺and CD8⁺ populations of CAR⁺ engineered T cells derived from the samesubject administered at a target ratio of approximately 1:1. Subjectswere administered a dose of CAR-expressing T cells (as a split dose ofthe CD4⁺ and CD8⁺ CAR-expressing T cells) at a single dose of dose level1 (DL1) containing 5×10⁷ CAR-expressing T cells. Beginning at three (3)days prior to CAR⁺ T cell infusion, subjects received a lymphodepletingchemotherapy with fludarabine (flu, 30 mg/m²) and cyclophosphamide (Cy,300 mg/m²).

Subjects were monitored for response and toxicities as described inExample 1. No CRS or neurotoxicity was observed in any of the subjects.Of the 4 subjects that were treated, two (2) subjects achieved PR (notdurable) and two (2) patients had progressive disease.

Example 3: Further Assessment of Response, Safety, Pharmacokinetics,Pharmacodynamics and Blood Analytes in Subjects with Relapsed andRefractory Non-Hodgkin's Lymphoma (NHL) After Administration ofAnti-CD19 CAR-Expressing Cells

Response outcomes, safety outcomes, pharmacokinetic and pharmacodynamicsparameters, and blood analytes were assessed in patients at a subsequentpoint in time in the clinical study described in Example 1 above.

A. Subjects and Treatment

The analysis at this time point presented in this example is based onassessment of a total of 91 subjects in the full DLBCL cohort (88 (65from the CORE cohort) assessed for response and 91 (67 from the COREcohort) assessed for safety) that had been administered the anti-CD19CAR-expressing cells. The FULL cohort included DLBCL, NOS de novo andtransformed from any indolent lymphoma, ECOG 0-2; the CORE cohort foranalysis included subjects having DLBCL, NOS and transformed fromfollicular lymphoma (tFL) or high grade B-cell lymphoma and with EasternCooperative Oncology Group performance status (ECOG PS) of 0 or 1.Approximately 90% of treated patients in the CORE cohort had at least 1poor-risk disease feature predictive of short median overall survival(OS) of 3-6 months, such as double/triple hit expressors, primaryrefractory disease, refractory to 2 or more lines of therapy, neverachieved CR, or never received autologous stem cell transplant (ASCT).In some embodiments a cohort of subjects having Diffuse large B-celllymphoma (DLBCL) not otherwise specified (NOS; de novo and transformedfrom follicular lymphoma tFL)) or high-grade B-cell lymphoma, with MYCand BCL2 and/or BCL6 rearrangements with DLBCL histology, and excludingsubjects with ECOG score of 2 or subjects who have received priorhematopoietic stem cell transplantation (HSCT), are administered CAR-Tcompositions as provided herein. In some embodiments, subjects of theCORE cohort are administered anti-CD19 CAR⁺ T cells at a single dose ofDL2 (1×10⁸ total CAR-expressing T cells).

At this time point, a total of 140 subjects had been leukapheresed, ofwhich 10 were awaiting manufactured composition, 2 had withdrawn beforemanufacturing, and 2 had compositions unavailable. Of another 18subjects whose products were available, 4 were awaiting treatment, 4 hadwithdrawn, and 10 had developed progressive disease or had died. A totalof 108 subjects had been administered the anti-CD19 CAR-expressingcells, of which 6 were not evaluable and 11 received non-conforminganti-CD19 CAR-expressing cells (compositions not necessarily meetingcertain specifications but deemed to be safe for administration).Subjects had received DL1 (n=45), double dose of DL1 (n=6) or DL2(n=40). Six (6) subjects with mantle cell lymphoma (MCL) had beenadministered CAR⁺ cells at DL1 (five treated with conforming product,one treated with non-conforming product), and five (5) had completed 28days of follow-up. One MCL subject had developed CRS, and none hadreceived tocilizumab or dexamethasone. Product had been available for98% of apheresed subjects (126/128) in the DLBCL cohort.

The subjects at this time-point included 5 patients that had beentreated in the outpatient setting (including four (4) subjects treatedwith DL1, one (1) treated with DL2; four (4) of which were included inthe CORE cohort). For subjects treated in the outpatient setting, medianage was 57 years old (range 26-61), 3 had DLBCL, NOS, 1 had tFL, and 1had PMBCL. All five (5) subjects had an ECOG scores of 0 or 1. Data onoutpatient results included results for three (3) additional subjectsthat had been treated in the outpatient setting (total of eight (8)subjects) and whose data became available after the time point for theanalysis in this Example.

The demographics and baseline characteristics of the full and corecohort subjects at the timepoint are set forth in Table E5.

TABLE E5 Patient Characteristics: DLBCL Cohort FULL CORE Characteristic(n = 91) (n = 67) Median Age, years (range), 61 (20-82) 60 (20-82) ≥65years, n (%) 34 (37) 24 (36) Male/Female, n (%) 61/30 (67/33) 46/21(69/31) B-NHL subtype, n (%) DLBCL, NOS de novo 59 (65) 51 (76)Transformed from FL (tFL) 19 (21) 16 (24) Transformed from MZL 6 (7)/4(4) 0 (tMZL)/CLL (tCLL) Follicular, Grade 3B/PMBCL 1 (1)/2 (2) 0Molecular subtype, n (%) Double/triple hit 18 (20) 16 (24) [High gradeB-cell lymphoma]^(a) Patient characteristics, n (%) Chemorefractory^(b)61 (67) 44 (66) ECOG PS 0-1/2 (pre-LD) 81 (89)/10 (11) 67 (100)/0 IPI3-5/Disease stage 3-4 38 (42)/70 (77) 24 (36)/49 (73) CNS involvement 2(2) 2 (3) Prior lines of therapy, 3 (1-12) 3 (1-8) median (range) Neverachieved CR 47 (52) 34 (51) Any HSCT 39 (43) 28 (42) Prior Autologous 36(40) 28 (42) Prior Allogeneic 5 (5) 0 HSCT, hematopoietic stem celltransplantation; LD, lymphodepletion. ^(a)At trial initiation, includedin DLBCL, NOS histology; based on most recent WHO criteria (Swerdlow etal., (2016) Blood 127(20): 2375-2390), are now considered “high-gradeB-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements with DLBCLhistology (double/triple hit). ^(b)SD or PD to lastchemotherapy-containing regimen or relapse <12 months after autologousSCT.

B. Safety and Response Outcomes after Treatment

As shown in Table E6, the objective response rate (ORR) was 74%,including 52% subjects who showed a complete response (CR). Theincidence of any grade of cytokine release syndrome (CRS) was 35%, with1% severe CRS; and the incidence of any grade of neurotoxicity (NT) was19%, with 1% severe NT.

TABLE E6 Response and Safety After CAR⁺ Cell Administration FULL COREAll Dose All Dose Levels Levels^(a) DL1S DL2S Best Overall Response 8865 34 27 (BOR), n^(b) ORR,% (95% CI) 74 (63, 83) 80(68, 89) 77 (59, 89)82 (62, 94) CR,% (95% CI) 52 (41, 63) 55(43, 68) 47 (30, 65) 63 (42, 81)Best Overall Response 88 65 34 27 (BOR), n^(b) Safety, n^(c) 91 67 34 29Any CRS, % (95% CI) 35 (25, 46) 36 (24, 48) 41 (25, 59) 24 (10, 44)sCRS(grade 3-4), % 1 (0, 6)  1 (0, 8)  38 (0, 15)   0 (95% CI) Any NT, %(95% CI) 19 (11, 28) 21 (12, 33) 24 (11, 41) 17 (6, 36)  sNT (grade3-4), % 12 (6, 21)  15 (7, 26)  21 (9, 38)  7 (1, 23) (95% CI) ^(a)Fourpatients treated on DL1D (dose level 1, two-dose schecule) with similaroutcomes. ^(b)Includes patients with event of PD, death, or 28-dayrestaging scans. One patient did not have restaging scans available.^(c)Includes all subjects who have received at least one dose ofconforming CAR-expressing cell product 28 days prior to data snapshotdate or died.

As shown in Table E7, high rates of response and low severe toxicity wasobserved in the full DLBCL population.

TABLE E7 Response After CAR⁺ Cell Administration By Diagnosis DLBCL,tCLL/ FL3B/ FULL NOS tFL MZL PMBCL BOR, n^(a) 88 57 19 10  2 ORR, % 7474 84 50 100 (95% CI) (63, 83) (60, 85) (60, 97) (19, 81) (16, 100) CR,% (95% CI) 52 51 63 30 100 (41, 63) (37, 64) (38, 84) (7, 65) (16, 100)Safety, n^(b) 91 59 19 10  3 Any CRS, 35 34 42 20 67 % (95% CI) (25, 46)(22, 47) (20, 67) (3, 56) (9, 99) sCRS (grade 3-4),  1  2  0  0  0 %(95% CI) (0, 6) (0, 9) Any NT, % 19 20 21 10  0 (95% CI) (11, 28) (11,33) (6, 46) (0, 45) sNT (grade 3-4), 12 14 11 10  0 % (95% CI) (6, 21)(6, 25) (1, 33) (0, 45) ^(a)Includes patients with event of PD, death,or 28-day restaging scans. One patient did not have restaging scansavailable. ^(b)Includes all subjects who have received at least one doseof conforming CAR⁺ expressing cells 28 days prior to data snapshot dateor died.

As shown in Table E8, high rate of response and a dose-dependentresponse was observed in the CORE cohort of subjects.

TABLE E8 Durable Response After CAR⁺ Cell Administration Dose Levels^(a)DL1S DL2S BOR, n^(b) 65 34 27 ORR (95% CI), % 80(68, 89) 77 (59, 89) 82(62, 94) CR (95% CI), % 55(43, 68) 47 (30, 65) 63 (42, 81) ≥3-mo f/u,n^(c) 52 29 19 3-mo ORR (95% CI), % 65 (51,78) 59 (39, 77) 74 (49, 91)3-mo CR (95% CI), % 54(40, 68) 41 (24, 61) 68 (43, 87) ≥6-mo f/u, n^(d)38 20 14 6-mo ORR (95% CI), % 47(31, 64) 40 (19, 64) 50 (23, 77) 6-mo CR(95% CI), % 42 (26, 59) 30 (12, 54) 50 (23, 77) ^(a)Four patients (CORE)treated on DL1D with similar outcomes. bIncludes patients with event ofPD, death, or 28-day restaging scans. One patient did not have restagingscans available. ^(c)The denominator is number of patients who receivedCAR⁺ cells ≥3 months ago, prior to data snapshot date, with an efficacyassessment at month 3 or prior assessment of PD or death. ^(d)Thedenominator is number of patients who received CAR⁺ cells ≥6 months ago,prior to data snapshot date, with an efficacy assessment at month 6 orprior assessment of PD or death.

Three-month objective response rates (ORR) among various subgroups ofsubjects in the poor-risk DLBCL subgroups, that included all DLBCLpatients treated at all dose levels in the core cohort, are shown inFIG. 22 . The results showed high durable ORR in the poor-risk DLBCLsubgroup.

Results for the duration of response (DOR) and overall survival (groupedby best overall response (non-responder, CR/PR, CR and/or PR)) are shownfor the full cohort and the core cohort cohorts of subjects, in FIGS.23A-23D. The results also showed 80% (16/20) of subjects with a CR at 3months stay in CR at 6 months, and 92% (11/12) of subjects with aresponse (CR or PR) at 6 months continue to show a response longer term.

FIG. 24 depicts the percentage of subjects at this timepoint who wereobserved to have experienced laboratory abnormalities andtreatment-emergent adverse events (TEAEs) (data for 5 patients with MCLtreated with conforming product at DL1 with at least 28 days offollow-up are not included). In addition to the TEAEs shown in FIG. 24 ,the following event terms were observed at Grade 3-4 in ≥5% of patients:encephalopathy (8%), Pancytopenia (5%) and Febrile neutropenia (7%).Eight patients (9%) had infusional toxicity, defined as AE on day ofadministration related to CAR⁺ cell administration, including flushing,headache, fever, pyrexia, chills, rigors, vomiting, rash, hives,pruritis, hypotension, wheezing, bronchospasm, shortness of breath,nausea, vomiting, back pain, cough, and infusion-related reaction.Events included chills (2), pyrexia (5), flushing (1), headache(1),hypotension (1), infusion related reaction (1), rash (1), pruritis(1), and vomiting (1), with 6 grade 1 events, 1 grade 2 (chills), and 1grade 3 (hypotension) event. TEAE in the core cohort did not differsubstantially from those in the full cohort. The most common relatedTEAEs in the subjects treated in the outpatient setting group were CRS,hypotension, vomiting, anemia, and dyspnea.

Table E9 sets forth the TEAEs and neurotoxicity that occurred in 25percent or more subjects in the FULL or CORE cohort, for subjects whoreceived DL1S and DL2S. No apparent dose-toxicity relationship wasobserved in the DLBCL population.

TABLE E9 TEAEs ≥25% in FULL cohort, CORE cohort, and CORE cohort by doselevel. FULL CORE^(a) CORE DL1S CORE DL2S Term, n (%) (N = 91) (n = 67)(n = 34) (n = 29) 85 (93) 63 (94) 33 (97) 26 (90) Anemia^(b) 64 (70) 48(72) 28 (82) 19 (66) Thrombocytopenia^(b) 48 (53) 41 (61) 20 (59) 19(66) Fatigue 34 (37) 25 (37) 11 (32) 12 (41) CRS 32 (35) 24 (36) 14 (41) 7 (24) Nausea 25 (27) 19 (28) 12 (35)  5 (17) Diarrhea 23 (25) 16 (24) 7 (21)  7 (24) ^(a)Includes 4 patients treated at dose level 1,two-dose schedule, ^(b)Laboratory anomalies.

FIG. 25 depicts the number and percentage of subjects that were observedto have CRS and/or NT at various time points after administration ofCAR⁺ cells. In this assessment, the median time to onset of first of CRSor NT event was observed to be 5 (range 1-14) or 10 (range 3-23) days,respectively. Within the first 72 hours after CAR⁺ cell administration,1 patient had NT (grade 1), and only 14% (13 of 91) had CRS (7 grade 1;6 grade 2). The median duration (Q1,Q3) of CRS or NT was 5 (4, 8) or10.5 (7, 19) days, respectively. NT was preceded by CRS in 12 of 17cases (71%). All evaluable NT events were resolved at the time ofanalysis except one grade 1 tremor and 2 patients died from progressivedisease with ongoing NT (based on safety database of reported eventsincluding additional subjects analyzed after the analysis timepointdescribed in this Example).

In the full cohort (n=91), selected subjects with onset of CRS or NTwere administered anti-cytokine therapy with tocilizumab and/ordexamethasone as follows: Tocilizumab alone, 4% (n=4); Dexamethasonealone, 9% (n=8); Tocilizumab and dexamethasone, 8% (n=7). The mediannumber of dexamethasone doses was 6 (range, 2-99); and the median numberof tocilizumab doses was 1 (range, 1-3).

Table E10 shows toxicity outcomes in subjects in the CORE cohort thatreceived a single dose at DL1 or DL2. No deaths occurred from CRS or NT.The median time to onset of CRS was 5 days (range, 2-14) and NT was 11.5days (range, 5-23). In the CORE cohort, 13% (n=9) received tocilizumab,and 18% (n=12) received dexamethasone to ameliorate toxicity. Eighteenpercent of subjects (12 of 67) exhibited neurotoxicity terms consistentwith encephalopathy, including encephalopathy (13%), 6% (4 of 67) hadaphasia and 3% (2 of 67) had seizures. In Table E10, the number ofsubjects or % of total subjects (parentheses) exhibiting an indicatedtoxicity outcome is shown at all dose levels or specifically in subjectsadministered DL1 or DL2. Also shown in brackets is the upper and lower95% confidence interval.

TABLE E10 Toxicity in Core Cohort Receiving Different Dose Levels. AllDose Levels^(a) DL1S DL2S n = 67 n = 34 n = 29 CRS, n (%) [95% CI] AnyGrade 24 (36) [24, 48] 14 (41) [25, 59] 7 (24) [10, 44] Grade 1/2 23(34) [23, 47] 13 (38) [22, 56] 7 (24) [10, 44] Grade 3/4 (sCRS) 1 (1)[0, 8]  1 (3) [0, 15] 0 Neurotoxicity^(b), n (%) [95% CI] Any Grade 14(21) [12, 33]  8 (24) [11, 41] 5 (17) [6, 36]  Grade 1/2  4 (6) [2, 15] 1 (3) [0, 15] 3 (10) [2, 27]  Grade 3/4 (sNT) 10 (15) [7, 26]  7 (21)[9, 38] 2 (7) [1, 23] Any, n (%) [95% CI] CRS or NT, n (%) 28 (42) [30,54] 15 (44) [27, 62] 10 (34) [18, 54]  sCRS or sNT, n (%) 10 (15) [7,26]  7 (21) [9, 38] 2 (7) [1, 23] ^(a)Four patients treated on DL1D withsimilar outcomes. ^(b)Includes confusional state, encephalopathy,aphasia, ataxia, cerebellar syndrome, delirium, depressed level ofconsciousness, dizziness, flat affect, hand-eye coordination impaired,memory impairment, tremor, agitation, disturbance in attention,dysarthria, mental status changes, muscular weakness, seizure,somnolence, and urinary incontinence.

Among twelve (12) subjects receiving nonconforming products, 10 at DL1and 2 at DL2, all had 28-day follow-up. CRS was observed in 33% of thesubjects (4/12), and NT was not observed in any of the subjects. Twosubjects received tocilizumab and 3 subjects received dexamethasone. Thetoxicity rates were comparable to those observed in the larger cohort ofsubjects administered conforming product. In the subjects receivingnonconforming products, pharmacokinetic (PK) expansion was higher insubjects with CRS/NT, subjects with high tumor burden or LDH levels.

C. Assessment of Outpatient Administration

Data for a total of eight (8) subjects were evaluated at this timepointthat had been treated in the outpatient setting (median age of 58.5 andECOG of 0 or 1) at multiple clinical sites, including 3 subjects whosedata was available subsequent to the time point analyzed for purposes ofthis Example. The mean length of hospitalization was 15.6 days forsubjects treated in the inpatient setting (SD 9.6, n=86) and 9.3 daysfor subjects treated in the outpatient setting (SD 11.9, n=8). A 40%reduction in length of hospitalization was observed in subjects treatedin the outpatient setting. The median number of days prior tohospitalization after outpatient CAR⁺ T cell administration was 5 days(range: 4-22). None required admission to the intensive care unit (ICU)after outpatient administration.

Among those of the 8 subjects treated in the outpatient setting withmore than 28-day post-administration follow-up, 1 remained outpatientthroughout the duration of the dose-limiting toxicity period. Seven (7)patients were admitted with fevers (1 on study day 4, the rest on studyday ≥5), 6 patients were admitted with CRS (4 grade 1, 2 grade 2) and 2patients with grade 1 NT. No patient experienced severe CRS or NT. One(1) patient was treated with tocilizumab without dexamethasone for CRS(grade 2), and no patients were treated with dexamethasone for CRS orNT. One patient was admitted 3 days after CAR⁺ T cell administration.

Among 91 subjects treated in the inpatient and outpatient settings, 11subjects (12%) required ICU admission for management of toxicity; 8subjects (9%) required ICU admission for management of CRS or NT; 2subjects (2%) required ICU admission for management of acute respiratoryevents (one related to CAR⁺ T cell administration, one unrelated). Six(6) subjects (6%) were intubated (based on safety database of reportedevents including additional subjects analyzed after the analysistimepoint described in this Example; n=94); 7 subjects (7%) receivedvasopressors (based on safety database of reported events, defined asexhibiting hypotension in the first 28 days after CAR⁺ T celladministration, in the TEAE assessment); and 2 subjects (2%) underwenthemofiltration (based on safety database of reported events). Theresults showed that very few patients required ICU-level care andassociated procedures. The results supported the feasibility ofoutpatient administration, with safe management of toxicity in theoutpatient setting, appropriate education and outpatient monitoring.

The assessment of outpatient administration supported the feasibility ofsafe outpatient administration. 30% of the subjects were notre-admitted.

D. Pharmacokinetic Assessment

Numbers of CAR⁺ T cells in peripheral blood and bone marrow at timepoints before administration (pre-treatment or pre-lymphodepletingchemotherapy (LDC)) and various time points post-treatment (with day ofadministration as day 1) in 87 subjects in the DLBCL cohort withevaluable PK, by flow cytometry using an antibody specific for thetruncated receptor used as a surrogate marker, and quantitativepolymerase chain reaction (qPCR) using primers specific for a woodchuckhepatitis virus post-transcriptional regulatory element (WPRE) presentin the vector encoding the chimeric antigen receptor (CAR). The areaunder the curve plotting numbers per microliter for the indicated CAR⁺cell population between days 0 and 28 (AUC₀₋₂₈) and the maximum or peakblood concentration of CAR⁺ cells (C_(max); CAR⁺ cells/μL blood) wereassessed. B-cell aplasia was assessed in peripheral blood by flowcytometry, by staining with CD19. Cytokines were measured using amultiplex cytokine assay. For safety analysis, the data from allsubjects receiving different dose levels were pooled. For responseanalysis, data were stratified by dose level. Statistical analysis wastwo-sided without multiplicity adjustment.

FIG. 9A shows detected numbers of CART cells per microliter of blood atvarious indicated time-points, as assessed by qPCR or flow cytometry.FIG. 9B shows CAR⁺ cells per microliter of blood versus microliter ofbone marrow at day 11±3. As shown in FIG. 9A, levels of CAR-expressingcells in samples from subjects were observed both by flowcytometry-based assays and qPCR-based assays. As shown in FIG. 9B, allsubjects (n=86 and 85 for flow cytometry and qPCR, respectively,excluding one patient that did not have flow cytometry results availableand 2 patients that did not have qPCR results available) with PK resultsassessed, showed detectable numbers of the CAR-expressing cells in theblood and bone marrow. Results were consistent with an observation thatCAR⁺ T cells had trafficked similarly to the bone marrow and blood.

Levels over time of CD4⁺ and CD8⁺ CAR-expressing cells (as assessed byAUC₀₋₂₈ and C_(max)) were compared in different patient subgroupsreceiving dose level 1 (DL1): diffuse large B-cell lymphoma de novo(DLBCL, NOS) or transformed from follicular lymphoma (tFL) (CORE; N=32),DLBCL transformed from marginal zone lymphoma or chronic lymphocyticleukemia (tMZL/tCLL; N=4), or mantle cell lymphoma (MCL; N=5), who hadreceived CAR-expressing T cells at DL1. As shown in FIGS. 10A and 10B,AUC₀₋₂₈ and C_(max), varied among subjects in different diseasesubgroups, with expansion of CD4⁺ and CD8⁺ CAR-expressing cells trendinglower in non-CORE subsets. PMBCL (n=2) and FL3B (n=1) not shown due tolimited patient numbers. Expansion in subjects receiving DL2 was similarto in subjects receiving DL1.

E. Pharmacokinetic Assessment by Dose Level

AUC₀₋₂₈ and C_(max) for CD3⁺, CD4⁺ and CD8⁺ CAR-expressing cells werealso compared for subjects having received dose level 1 (DL1; n=32) andthose having received dose level 2 (DL2; n=27), in the CORE cohort(subjects with DLBCL, NOS or high grade B-cell lymphoma (double/triplehit); N=59). As shown in FIGS. 11A and 11B and in Table E1l, a highermedian AUC₀₋₂₈ was observed for CD3⁺, CD4⁺ and CD8⁺ CAR-expressing cellswas observed in subjects that received DL2, compared to subjects who hadreceived DL1. Similarly, a trend of higher expansion in subjects who hadreceived DL2 was observed in the full DLBCL cohort. A higher durabilityof response (DOR) at 3 months also was observed among subjects who hadreceived DL2 as compared to those having received DL1, without anincrease in toxicity. The median time to C_(max) (T_(max)) for CD4⁺ andCD8⁺ CAR⁺ cells was similar between subjects who received DL1 and DL2.

Increased CAR⁺ T cell exposure was observed in DL2 versus DL1,corresponding to an increased durability of response without increasedtoxicity in DL2 subjects.

TABLE E11 Pharmacokinetics in Subjects Grouped by Dose Levels in Corecohort DL1S DL2S Total, DL1S and (n = 32) (n = 27) DL2S (n = 59) CD3⁺Cmax, median 48.2 96.2 65.8 (cells/μL) Q1, Q3 15.6, 151.3 30.2, 219.519.0, 204.2 Min, max 0.1, 7726.3 1.1, 1280.9 0.1, 7726.3 T_(max), median(days) 14.5 15.0 15.0 Q1, Q3 11, 15 11, 15 11, 15 Min, max 9, 24 8, 318, 31 AUC₀₋₂₈, median 477.7 823.1 542.4 (cells * day/μL) Q1, Q3 165.9,999.3 155.8, 3628.3 155.8, 3381.9 Min, max 1.8, 142816.7 16.5, 16087.81.8, 142816.7 CD4⁺ C_(max), median 7.0 14.9 7.7 (cells/μL) Q1, Q3 2.6,46.0 2.0, 46.8 2.5, 46.8 Min, max 0.1, 3039.9 0.2, 169.4 0.1, 3039.9T_(max), median (days) 14.0 15.0 15.0 Q1, Q3 11, 15 11, 15 11, 15 Min,max 8, 24 8, 31 8, 31 AUC₀₋₂₈, median 71.1 166.1 91.5 (cells * day/μL)Q1, Q3 26.4, 274.7 18.1, 679.0 23.9, 368.8 Min, max 1.2, 68990.3 2.9,4266.8 1.2, 68990.3 CD8⁺ C_(max), median 26.1 62.8 43.6 (cells/uL) Q1,Q3 3.7, 111.2 26.2, 171.7 9.1, 151.6 Min, max 0.0, 5237.6 0.7, 1261.80.0, 5237.6 T_(max), median (days) 15.0 15.0 15.0 Q1, Q3 11, 16 11, 1711, 16 Min, max 4, 28 8, 31 4, 31 AUC₀₋₂₈, median 347.2 606.6 412.2(cells * day/μL) Q1, Q3 52.1, 871.4 155.7, 2463.4 72.1, 1852.5 Min, max0.3, 81865.9 4.7, 15570.0 0.3, 81865.9

F. Persistence

Persistence of CAR-expressing cells and CD19⁺ B cell aplasia (lownumbers or absence of CD19⁺ B cells) was assessed at various time pointsin evaluable subjects with DLBCL that had been administered CAR⁺ Tcells, based on detectable CD3⁺, CD4⁺ or CD8⁺ CAR-expressing cell levelsand levels of CD19⁺ B-cells detected in the blood, respectively. Theresults are set forth in Table E12. Among subjects evaluated atprogression (time of progression regardless of BOR; n=37), a median of0.17 CD4⁺ CAR⁺ cells/μL (range, 0-65.5 cells/μL) and a median of 0.15CD8⁺ CAR⁺ cells/μL (range, 0-131.8 cells/μL) were observed atprogression. Among subjects evaluated at relapse (at the time ofprogression after achieving CR) (n=12), a median of 0.17/μL (range,0-35.1 cells/μL) CD4⁺ CAR-expressing cells and a median of 0.20 cells/μL(range, 0-131.8 cells/μL) CD8⁺ CAR-expressing cells were observed atrelapse Long-term persistence of CAR-expressing cells was observed in75% of evaluable subjects with DLBCL at 12 months. Long-term persistenceof B cell aplasia also was observed in 75% of the subjects at 12 months,and in subjects regardless of relapse status. The results are consistentwith a conclusion that the anti-CD19 CAR-expressing cells exhibitedlong-term persistence in most subjects, and suggest the potential forongoing, low-level disease control even in relapsed patients.

Of subjects who relapsed, 91.7% (11/12) had detectable CAR-expressingcells in the blood at the time of relapse. This result is consistentwith a conclusion that a combination therapy or other intervention insome embodiments may be used to augment and/or boost CAR-expressingcells such as those that may be exhausted.

TABLE E12 CAR⁺ Cell Long-Term Persistence and CD19 Aplasia Month MonthMonth Month At At 3 6 9 12 Progression Relapse CAR T persistence in 5030 18 12 37 12 evaluable patients, n CD3⁺, % 100 80.0 77.8 75.0 91.991.7 CD4⁺, % 88.0 63.3 50.0 41.7 83.8 83.3 CD8⁺, % 90.0 70.0 55.6 50.083.8 75.0 CD19⁺ 96.0 93.3 77.8 75.0 97.3 100 B-cell aplasia (<1cell/μL), %

G Pharmacokinetic Assessment and Toxicity

AUC₀₋₂₈ and C_(max) of CD4⁺ and CD8⁺ CAR-expressing cells was alsocompared for subjects in the core cohort with any grade (in thisassessment, any of grade 1-4; no grade 5 CRS or NT observed) cytokinerelease syndrome (CRS) or neurotoxicity (NT) to subjects that were notassessed as exhibiting any grade of CRS or NT. The median CD4⁺ CAR⁺AUC₀₋₂₈ (Q1, Q3) was 59 (18, 210) for no CRS (grade 0; n=43), and 267(91, 1510) for any CRS (grades 1-4; n=20) (p=0.001); the median CD8⁺CAR⁺ AUC_(0.28) (Q1, Q3) was 310 (36, 900) for no CRS (grade 0; n=43),and 605 (174, 5619) for any CRS (grades 1-4; n=20) (p=0.021); the medianCD4⁺ CAR⁺ AUC₀₋₂₈ (Q1, Q3) was 71 (23, 244) for no NT (grade 0; n=50),and 1269 (184, 3057) for any NT (grades 1-4; n=13) (p=0.003); the medianCD8⁺ CAR⁺ AUC_(0.28) (Q1, Q3) was 304 (43, 799) for no NT (grade 0;n=50), and 2463 (607, 7691) for any NT (grades 1-4; n=13) (p=0.004). Asdescribed above and shown in FIGS. 12A-12D, higher CD4⁺ and CD8⁺CAR-expressing cell levels over time were associated with CRS and NT.

H. Pharmacokinetic Assessment and Response

The number of peak CD3⁺ CAR⁺ (CD3⁺ C_(max)) was assessed over time insubjects who had a best overall response (BOR) of CR, PR or PD. As shownin FIGS. 13A and 13B, a trend towards better BOR was observed insubjects with higher expansion, with variability among subjects.

I. Pharmacokinetic Assessment by Blood Analytes and Patient Parameters

Pre-CAR⁺ T cell treatment (pre-lymphodepleting chemotherapy) plasmacytokine levels, including interleukin-7 (IL-7), IL-15, macrophageinflammatory protein (MIP-1a), were assessed in subjects that exhibiteda CAR⁺CD3⁺ blood C_(max)>500 CAR⁺ T (N=55) as compared to in subjectsthat exhibited CAR⁺CD3⁺ blood C_(max)<500 CAR⁺ T (N=7). As shown in FIG.14A, elevated pre-CAR⁺ T cell treatment cytokine plasma levels wereobserved to be associated with CAR⁺CD3⁺ C_(max)>500 CAR⁺ T (Wilcoxon Pvalues <0.05 (without multiplicity of adjustment); except for IL-7p=0.07).

Peak levels of various plasma cytokines (IL-6, IL-10, IL-16, interferongamma (IFN-γ), tumor necrosis factor alpha (TNF-α), MIP-1α, Monocytechemoattractant protein-1 (MCP-1), and C-X-C motif chemokine 10(CXCL10)) were also assessed in subjects that exhibited CAR⁺ CD3⁺ bloodC_(max)>500 CAR⁺ T (N=68) as compared to subjects that exhibited CAR⁺CD3⁺ blood C_(max)<500 CAR⁺ T cells/μL; N=9). As shown in FIG. 14B,higher peak cytokine levels were observed to be associated withCAR⁺CD3+C_(max)>500 CAR⁺ T cell/μL (Wilcoxon P values <0.05; withoutmultiplicity of adjustment).

Relationship between pre-CAR⁺ T cell treatment (pre-lymphodepletingchemotherapy (LDC)) volumetric tumor measurement sum of productdimensions (SPD), as an indicator of tumor burden, and AUC₀₋₂₈ of CD3⁺CAR⁺ T cells, representing CAR⁺ T exposure over time, was assessed. Asshown in FIG. 15 , a positive correlation was observed between baselineSPD and CD3⁺ AUC₀₋₂₈, with a Spearman correlation of 0.32 and p=0.019. IPre-treatment patient parameters and response and toxicity outcomes

Pre-CAR⁺ T cell treatment (pre-LDC) analyte levels, including cytokinesand inflammatory markers such as Ferritin, C-reactive protein (CRP),D-dimer (fibrin degradation product), IL-6, IL-10, IL-15, IL-16 TNF-α,MIP-1α, and MIP-1β, were compared for subjects with any grade (here,grade 1-4) cytokine release syndrome (CRS) or neurotoxicity (NT) tosubjects that did not have any CRS or NT (grade 0). In this cohort,among subjects with CRS grade 1-4, all but one CRS events weredetermined to be grade 1 or 2. As shown in FIG. 16A (CRS) and FIG. 16B(NT), higher peak plasma cytokine levels and inflammatory marker levelswere observed to be associated with CRS and NT, based on univariateanalysis (Wilcoxon P values <0.05 for all analytes except ferritin forCRS (p=0.14) and CRP for CRS (p=0.09)). For CRS, after adjusting tumorburden in a multivariable analysis, MIP-1β, IL-10 and TNF had p<0.05;for NT, IL-15, IL-6, MIP-1α, and TNF had p<0.05.

Pre-treatment (pre-LDC) patient parameters, such as levels of lactatedehydrogenase (LDH) and a volumetric tumor measurement such as sum ofproduct dimensions (SPD), as an indicator of tumor burden, were comparedbetween subjects that were not observed to have developed CRS orneurotoxicity versus subjects that were observed to have developed CRSor NT. As shown in FIG. 17 , subjects with CRS or NT exhibited higherlevels of pre-treatment patient parameters such as SPD (cm²) and LDH(U/L) levels; such levels were observed to be correlated with CRS or NT,with univariate statistical analysis. Other patient parameters that wereobserved to be associated with CRS and NT include shorter time sincediagnosis (p=0.05 and p=0.09, for CRS and NT, respectively). Patientparameters that were observed not to be associated with CRS or NTincluded age (p=0.19 and p=0.54, respectively) and prior numbers oftherapies (p=0.67 and p=0.59, respectively), disease stage 0-2 vs 3-4(p=0.79, p=0.51), and patient weight (p=0.35 and p=0.44, respectively).

FIG. 18A shows pre-treatment SPD and LDH levels among individualpatients (dots; with shading of individual dots indicating whether theindividual patients did or did and did not exhibit any gradeneurotoxicity (left-hand panel) or did or did not exhibit any grade CRS(right-hand panel). In FIG. 18A, dotted lines on the y and x axesdelineate SPD≥50 cm² and LDH ≥500 U/L, respectively. As shown in FIG.18A, an SPD of approximately 50 cm² or higher, and/or an LDH ofapproximately 500 U/L or higher, were observed to be associated withrisk of NT and CRS. Calculated odds ratio estimates for developing CRSor NT in subjects above or below the SPD and LDH levels indicated bydotted lines in FIG. 18A, with 95% confidence intervals (CI), aredepicted in FIGS. 18B and 18C. An odds ratio over 1 indicated anincreased probability or likelihood of developing CRS or NT. As shown,SPD of 50 cm² or higher, and LDH of 500 U/L or higher, were observed tobe associated with increased risk of developing CRS or NT. SPD of 50 cm²or higher and LDH of 500 U/L or higher was observed to be associatedwith an approximately 8-fold increased risk in developing any grade CRSand NT, and SPD of lower than 50 cm² and LDH of lower than 500 U/Lshowed a reduced risk of any grade CRS and NT. The results wereconsistent with an association of baseline patient parameters, includinghigh tumor burden and inflammatory biomarkers, with CAR⁺ T cellexpansion and increased rates of CRS and neurotoxicity.

Various pre-treatment (pre-LDC) patient parameters, including markersassociated with tumor burden (SPD), inflammatory cytokines and otherblood analytes, including LDH, ferritin, CRP, D-dimer, SAA-1, IL-6,IL-10, IL-15, IL-16, TNF-α, IFN-γ, MIP-1α and CXCL10, were compared forsubjects with and without a durable response at 3 months, withunivariate statistical analysis. As shown in FIG. 19 , certain markersof tumor burden, markers of inflammation or inflammatory cytokines wereobserved to be lower in subjects that exhibited a durable response (pvalue <0.05 for all parameters except SPD (p=0.1274)). Similar resultswere observed in subjects receiving DL2, when analyzed alone. An inverseassociation of baseline patient parameters, including high tumor burdenand inflammatory biomarkers, with durable response was observed. In someaspects, such inverse association may be due to higher expansion andexhaustion of CAR⁺ T cells.

Relationships between patient factors, clinical correlates and bloodanalytes to developing of degrees of CRS and NT were assessed usingstatistical analysis based on univariate nonparametric tests. Table E13lists the results of the univariate analysis. In this assessment, age<40 years and no prior HSCT correlated with incidence of CRS or NT.Subjects with age <40 years were not observed to have statisticallydifferent rates of higher tumor burden than older patients. Subjectswith ECOG score of 2 did not have statistically different rates ofhigher tumor burden compared to subjects with ECOG score 0-1. Thosewithout prior HSCT or double/triple hit or double expressor were notassociated with CRS or

NT.

TABLE E13 Univariate Analysis of Key Subgroups CRS NT Variable, AnyGrade Grade Any Grade Grade n (%) Grade 1/2 3/4 Grade 1/2 3/4 FULLPopulation (N = 91) Age <40 years  5 (63)  4 (50)  1 (13) 3 (38) 0  3(38) (n = 8) 40-64 years 19 (39) 19 (39) 0 9 (18) 5 (10) 4 (8) (n = 49)≥65 years  8 (24)  8 (24) 0 5 (15) 1 (3)   4 (12) (n = 34) Pre-LD ECOGPS 0-1 28 (35) 27 (33) 1 (1) 15 (19)  4 (5)  11 (14) (n = 81) 2  4 (40) 4 (40) 0 2 (20) 2 (20) 0 (n = 10) Double/triple hit or double expressorYes 12 (40) 12 (40) 0 6 (20) 3 (10)  3 (10) (n = 30) No  6 (27)  6 (27)0 4 (18) 2 (9)  2 (9) (n = 22) Prior HSCT Yes 10 (26) 10 (26) 0 5 (13) 4(10) 1 (3) (n = 39) No 22 (42) 21 (40) 1 (2) 12 (23)  2 (4)  10 (19) (n= 52)

K Peak Blood Analytes, Response and Toxicity

Peak post-treatment plasma levels of blood analytes, including cytokinesand inflammatory markers such as CRP, Serum Amyloid A1 (SAA-1), IL-2,IL-6, IL-10, IL-15, TNF-α, MIP-1α, MIP-1β, MCP-1, CXCL10 and C-C MotifChemokine Ligand 13 (CCL13) were compared for subjects with grade 1-4cytokine release syndrome (CRS) or neurotoxicity (NT) to subjects thatwere not observed to have any CRS or NT. As shown in FIG. 20A (CRS; CRSgrade 0, n=51; CRS grades 1-4, n=28) and FIG. 20B (NT; NT grade 0, n=63;NT grades 1-4, n=16), higher peak plasma cytokine levels andinflammatory marker levels were observed to be associated with CRS andNT (Wilcoxon P values <0.001 for no CRS vs. any CRS and for no NT vs.any NT, except IL-15 (P=0.05 and 0.006, respectively)).

Peak plasma levels of blood analytes, including cytokines andinflammatory markers such as CRP, SAA-1, IL-5, IL-6, IL-7, IL-8, IL-15,Lymphotoxin-alpha (LT-α), TNF-α, IFN-γ, MIP-1α, MIP-1β, MCP-1, CXCL10,and Transforming growth factor beta (TGF-β), were assessed for subjectswith a best overall response (BOR) of complete response (CR) or partialresponse (PR) (N=57) compared to levels in subjects with stable disease(SD) or progressive disease (PD) (N=17); or for subjects with a 3-monthSD or PD (SD/PD) (N=31), compared to subjects who exhibited CR/PR at3-months (N=35). As shown in FIG. 21A (best overall response (BOR)) andFIG. 21B (month 3 response), lower peak plasma cytokine levels andinflammatory marker levels were observed to be associated with betterBOR and response at month 3 (Wilcoxon P values <0.05 withoutmultiplicity of adjustment).

In this study, administration of the anti-CD19 CAR⁺ cell compositionswas administered to subjects with relapsed/refractory aggressivenon-Hodgkin lymphoma (NHL) that have poor-risk disease features.Responses, including durable responses, were observed, including 81%ORR, 63% CR at DL2, with 80% of patients in CR at 3 months remaining inCR at 6 months at all dose levels, median DOR of subjects treated at alldose levels of 9.2 months, with medium duration of CR not having beenreached at the time point of analysis in this example. The results alsowere consistent with manageable toxicity levels and a favorable safetyprofile that in some embodiments may be consistent with outpatientadministration. Low rates of severe CRS (1%) and severe neurotoxicity(12%) were observed, with few events in first 72 hours. Results wereconsistent with feasibility of outpatient administration.

Pharmacokinetic assessments showed that higher expansion of CAR⁺ T cellswas generally associated with increased rates of CRS and NT. Subjectsreceiving DL2 showed higher CAR T exposure compared to subjectsreceiving DL2, which generally corresponded to increased durability ofresponse without increased incidence of toxicity. In some aspects,pre-treatment, such as pre-LDC, patient factors, including homeostaticand inflammatory cytokines and tumor burden, were observed to beassociated with and/or drive very high expansion and toxicity. Theadministered CAR⁺ T cells were shown to expand in the blood and bonemarrow of all patients, with variability among subjects and betweendisease types. The administered CAR⁺ T cells also exhibited long-termpersistence, with 75% (9/12) of evaluable patients having detectable CART cells at 12 months. CAR T cells and B cell aplasia were observed to bestill present at time of relapse (11/12 and 12/12 patients,respectively), supporting that tumors may evade CAR T cell action andthat combination strategies may be effective to prevent relapse oraugment, boost or enhance exhausted CAR T cells. In general, a trend ofhigher response was observed with higher expansion, with variabilityamong subjects, supporting that other patient factors and/or diseasecharacteristics, e.g., tumor burden, may be contribute to determiningresponse.

Example 4: Attributes of Therapeutic T Cell Composition forAdministration and Process for Generation of Composition

Exemplary therapeutic T cell compositions containing autologous T cellsexpressing a chimeric antigen-receptor (CAR) specific for CD19, used foradministration in Examples 1 and 2 above were assessed for greater thanone hundred phenotypic, functional, and cell health related attributes,using flow cytometry and in vitro assays. Therapeutic cell compositionsgenerated for subjects enrolled in a clinical study evaluating anti-CD19CAR-T cell therapy for treatment of relapsed/refractory B-cellnon-Hodgkin lymphoma were examined (N=63; core cohort). Cells wereassessed before and after engineering, for various attributes. Exemplaryattributes that were assessed are set forth in Table E14. Memory andcell health phenotypes of the CAR T cells were examined using flowcytometry. T cell functionality was assessed using in vitroantigen-specific bioassays. Characterization and release testing wasconducted on therapeutic T cell compositions that had undergone arepresentative number of freeze-thaw cycles.

TABLE E14 Representative characterization attributes measured intherapeutic cell compositions containing anti-CD19 CAR T cells CellComposition Characterization Class Representative Attribute Cell HealthViability Active intracellular caspase-3 Annexin V Memory phenotype CCR7(C-C chemokine receptor type 7) Cell function Inflammatory cytokinessuch as TNF-α (tumor necrosis factor α)

For generation of cell compositions for administration, autologous cellswere isolated from the subjects via leukapheresis. Leukapheresis sampleswere subjected to a process for generation of CAR-expressing cells. Theprocess involved washing of cells using an automated wash andimmunoaffinity based selection for purification of CD4⁺ and CD8⁺ Tcells, resulting in two compositions, enriched for CD8⁺ (in which amedian of 99%, Inter Quartile Range (IQR) 98-100%, of cells were CD8⁺)and CD4⁺ (in which a median of 99%, IQR 99-100%, cells were CD4⁺) cells,respectively.

Cells of the enriched CD4⁺ and CD8⁺ compositions were separatelysubjected to lentiviral transduction with a vector encoding an anti-CD19CAR with a 41BB costimulatory domain. Transduced populations then wereseparately incubated in the presence of stimulating reagents for cellexpansion. Expanded CD8⁺ and CD4⁺ cells were formulated andcryopreserved separately and stored prior to administration. To minimizevariations, between lots and/or cell compositions derived from differentpatients, such as those having different patient attributes, inparameters indicative of cell health, cells were held at constantvolumes across lots. Cell products exhibited a tight range of viablecell concentrations (based on an assessment of cell compositions for onegroup of subjects, CD8⁺: median 31×10⁶ cells/mL, IQR 28-40×10⁶ cells/mL,N=38; CD4⁺: median 35×10⁶ cells/mL, IQR 31-40×10⁶, N=36).

As shown in FIG. 26 and summarized in Table E15, the automated T cellpurification resulted in pure CD8⁺ and CD4⁺ T cell populations. Thisstrategy reduced the probability of transducing non-T cells and resultedin high T cell purities in therapeutic cell composition independent ofthe expansion duration.

TABLE E15 T cell purity (% of total leukocytes) by process step DrugLeuka- Post- Mid- Compo- pheresis Purification Process sition CD4⁺ TCell Median 17.6 95.3 98.9 99.2 frequency IQR 11.7-24.7 92.1-98.598.1-99.2 98.7-99.6 CD8⁺ T Cell Median 19.2 96.0 98.9 99.3 frequency IQR13.2-30.9 93.4-98.6 98.0-99.2 98.4-99.7

At the site of administration, cell compositions were thawed andadministered separately, according to a target volume of eachcomposition corresponding to the number of CD8⁺ CAR⁺ and CD4⁺ CAR⁺ cellsin the appropriate dose (such as for DL1, containing 5×10⁷ totalCAR-expressing T cells (2.5×10⁷ each of CAR-expressing CD4⁺ andCAR-expressing CD8⁺ cells), or DL2, containing 1×10⁸ totalCAR-expressing T cells (5×10⁷ each of CAR-expressing CD4⁺ andCAR-expressing CD8⁺ cells)).

During the clinical trial there was a process change from a high-volumeformulation to low-volume formulation. The post change therapeutic cellcomposition was formulated at a constant low volume, with a tightlycontrolled range of viable cell concentrations. In some cases, alow-volume formulation was used instead of a high-volume formulation.Parameters indicative of health of the CAR-expressing T cells in thecompositions for administration were assessed, such as by measuring,post-thaw, viability, cell surface Annexin V expression and levels ofactive intracellular Caspase 3, in cell compositions that wereformulated with high volume and low volume.

The process change from the high-volume formulation to the low-volumeformulation resulted in increased process robustness and decreasedvariability of the cell health attributes. Values for concentration,percentage of viable cells, and percentage of active caspase-3 negativecells among CD4⁺ and CD8⁺ T cells from individual cell compositions areshown in FIGS. 27A-27C and are summarized in Table E16. The medianpercentage of Annexin V-expressing cells was 11% (IQR 9-18%; N=33) ofCD8⁺ CAR⁺ T cells and 10% (IQR 8-17%; N=31) of CD4⁺CAR⁺ T cells. Caspase3 expression was observed to be similar to Annexin V expression. Theshift to low-volume formulation resulted in increased robustness of theprocess and reduced the variance of cell health attributes.

TABLE E16 Cell Health Attributes CD4⁺ CD8⁺ High Low High Low FormulationFormulation Formulation Formulation Volume Volume Volume Volume CellConcentration Median 17.1 37.2 12.1 30.8 ×10⁶ cells/mL IQR 15.9-19.331.7-40.4 10.9-15.3 28.5-38.0 Cell viability % Median 82.8 82.5 72.080.3 IQR 79.5-84.7 80.4-84.3 69.3-76.6 76.4-83.3 % Caspase-3 Median 82.882.5 negative cells IQR 79.5-84.7 80.4-84.3

The quantities of CAR⁺CD4+ and CAR⁺CD8⁺ T cells in the composition foradministration were precisely controlled. The number of cells actuallyadministered to an exemplary set of subjects was observed to be within8% or less of the target number of cells for a given dose:

-   -   2.4-2.7×10⁷ (target ±8%) CD4⁺CAR⁺ T cells and 2.4-2.7×10⁷        (target ±8%) CD8⁺ CAR⁺ T cells for subjects administered cells        at DL1 (n=48)    -   4.6-5.1×10⁷ (target ±8%) CD4⁺CAR⁺ T cells or 4.6-5.1×10⁷ (target        ±8%) CD8⁺ CAR⁺ T cells for subjects administered cells at DL2        (n=20).

The range of administered dose was found to have low variability in adifferent exemplary set of subjects:

-   -   48-52×10⁶ CD3⁺ CART cell at DL1 (n=34)    -   96-101×10⁶ CD3⁺ CAR⁺ T cells at DL2 (n=29)    -   24-27×10⁶ CD4⁺CAR⁺ or CD8⁺ CAR⁺ T cells at DL1 (n=34)    -   46-51×10⁶ CD4⁺ CAR⁺ or CD8⁺ CAR⁺ T cells at DL2 (n=29).

As shown in FIG. 28A, CAR-expressing T cell compositions administered tosubjects were observed to exhibit high T cell purity and low variancebetween lots. In view, e.g., of process and product controls,therapeutic cell compositions containing CAR T cells were observed tohave low lot-to-lot variability in cell-specific T cell function.

In vitro antigen-specific cytokine accumulation and intracellularcytokine staining (ICS) showed a similar low variance between lots forcytokine production for multiple cytokines (IL-2, TNF-α and IFN-γ). Inan exemplary ICS experiment, cells from compositions were stimulatedwith CD19, stained for cytokines, including TNF-α, and surface proteins,including C-C chemokine receptor type 7 (CCR7) as a memory phenotypemarker, and analyzed by flow cytometry. The number of cells in thecomposition for administration that were positive for the cytokines orsurface proteins were determined. FIG. 28B shows the number of CD4⁺CAR⁺and CD8⁺CAR⁺ cells, CD4⁺CAR⁺ TNF-α⁺ and CD8⁺ CAR⁺NF-α⁺ cells,CD4⁺CAR⁺CCR7⁺ and CD8⁺CAR⁺CCR7⁺ and present in CAR T cell compositionsfor administration at DL1 and DL2. These results are summarized in TableE17. The results show low variability in the number of CD4⁺CAR⁺ andCD8⁺CAR⁺ cells, CD4⁺CAR⁺TNF-α⁺ and CD8⁺ CAR⁺TNF-α⁺ cells, CD4⁺CAR⁺CCR7⁺and CD8⁺ CAR⁺CCR7⁺ cells. For example, a tight range for the number ofcells positive for TNF-α production was observed (n=61).

TABLE E17 Controlled dose, T cell phenotypes, and cell specific function(×10⁶ cells) CD4⁺CAR⁺ CD8⁺CAR⁺ CD4⁺CAR⁺ CD8⁺CAR⁺ CD4⁺CAR⁺ CD8⁺CAR⁺TNF-α⁺ TNF-α⁺ CCR7⁺ CCR7⁺ DL1 DL2 DL1 DL2 DL1 DL2 DL1 DL2 DL1 DL2 DL1DL2 Median 25.1 50.1 25.0 50.0 23.0 47.4 19.9 41.7 11.1 15.8 8.9 15.0IQR 24.7-25.3 49.6-50.0 24.8-25.2 49.6-50.4 21.3-23.5 46.6-48.518.3-21.8 39.9-46.0 5.5-14.1 11.0-26.0 3.0-14.8 8.6-25.3

A parameter indicative of production by CAR⁺ cells of tumor necrosisfactor alpha (TNFα) after stimulation with CD19 showed a narrow rangeamong different lots, with relative standard deviation (RSD) of 37% forCD4⁺CAR⁺ T cells (N=59) and 51% for CD8⁺ CAR⁺ T cells (N=61).

The results were consistent with an observation that a composition thatcontains a precise and consistent dose of CD4⁺ and CD8⁺ CAR T cells,control and optimization of CD4⁺ and CD8⁺ T cell culture conditions, lowvariability of cytokine production, and/or constant formulation andvolume of the composition for administration can lead to consistent cellhealth in the composition. In provided embodiments, aspects of suchmanufacturing and control process contribute to low variability inattributes of such cell compositions engineered using cells from, andgenerated for administration to, a number of different subjects. Suchaspects in some aspects include the use of a precise, consistent flatdose of administered CD4⁺ and CD8⁺ cells among subjects; control andoptimization of CD4⁺ and CD8⁺ T cell culture conditions such as thosethat result in low between-drug product lot variability of phenotypes(e.g., CCR7) and in vitro function (e.g., IL-2, TNF-α and IFN-γproduction after antigen stimulation) such as among different subjects;and the use of constant formulation and volume of drug product which canresult in or contribute to consistency among therapeutic cellcompositions generated by the method in attributes indicative of cellhealth.

Example 5: Biomarker Assessment in Pre- and Post-administration TumorBiopsies from Subjects with Relapsed and Refractory Non-Hodgkin'sLymphoma (NHL) for Administration of Anti-CD19 CAR-Expressing Cells

Expression of several biomarkers was assessed in tumor biopsiescollected from subjects before and/or after administration ofCAR-expressing cells.

A. Tumor Biopsy Samples

Tumor biopsies were collected from selected subjects with relapsed orrefractory (R/R) diffuse large B-cell lymphoma (DLBCL) or mantle celllymphoma (MCL) who received treatment with therapeutic CAR⁺ T cellcompositions containing autologous T cells expressing a chimericantigen-receptor (CAR) specific for CD19, described above in Examples 1and 2 above, based on the time point of assessment in Example 1.A.2.Tumor biopsies were obtained prior to administration of the CAR⁺ T cells(pre-treatment) and at 7 to 20 days after administration(post-treatment). Results are described in this example for evaluationthrough the time-point in Example 1.A.2, in an ongoing study. Resultsfrom 43 biopsies (26 pre-treatment; 17 post-treatment and 15 matchedpairs) from 28 total subjects (25 DLBCL and 3 MCL) were examined.

B. Assessment of Biomarkers, Response and Safety Outcomes

Infiltration of CAR⁺ T cell in the tumor biopsy was quantified using insitu hybridization (ISH) probes specific to the mRNA encoding theanti-CD19 CAR. CAR⁺ T cells, non-CAR T cells and B cells were enumeratedusing multiplex immunofluorescence (IF) assays detecting for a cellsurface surrogate marker for CAR-expressing cells, CD4, CD8, CD19, CD20,CD73, FOXP3, CD163, IDO and PD-L1. Tumor biopsy sections were stainedwith hematoxylin and eosin (H&E) and assessed for tissue quality andtumor identification. Immunofluorescence images were analyzed using animage analysis software. Potential correlations to response outcomeswere assessed using statistical analysis based on univariate t-tests,and the p-values were 2-sided without multiplicity adjustment.

Subjects were assessed for response and safety outcomes, including byassessing the tumor burden at various time points after administrationof the CAR⁺ T cells, including at 3 months after administration, anddetermining whether the subject had progressive disease (PD), stabledisease (SD), partial response (PR), or complete response (CR). Safetyoutcomes evaluated included neurotoxicity (neurological complicationsincluding symptoms of confusion, aphasia, encephalopathy, myoclonusseizures, convulsions, lethargy, and/or altered mental status), gradedon a 1-5 scale, according to the National Cancer Institute—CommonToxicity Criteria (CTCAE) scale, version 4.03 (NCI-CTCAE v4.03).

C. Results

The observed objective response rate (ORR; including CR and PR) was 71%(20/28) in the subjects for which biopsies were assessed. Grade 1, 2 CRSwas observed in 36% (10/28; grade 1, 2) of the subjects for whichbiopsies were assessed, and Grades 2-4 NT was observed in 18% (5/28) ofthe subjects for which biopsies were assessed.

Pre-treatment tumor biopsies were observed to contain varying cellularcompositions: tumor cells (median: 77%; range 5-96%), CD4⁺ cells (0.90%;0.02-15%), and CD8⁺ cells (1.5%; 0-23%). The results showed thatsubjects with a CR or PR at 3 months after CAR⁺ T cell administrationhad a higher percentage of endogenous CD4⁺ cells in pre-treatment tumorscompared those with a PD (CR, PR median: 7.9%; PD median: 0.38%;p<0.0001). Percentages of CD8⁺ cells in pre-treatment tumors did notdiffer between the 3 month response groups (CR, PR median: 1.9%; PDmedian: 0.47%; p=0.6496).

In the post-treatment biopsies, CAR⁺ T cell were observed to haveinfiltrated the tumor, and constituted up to 22% of cells in the biopsysample. The level of tumor infiltration in post-treatment samples (7 to20 days after administration) was observed to be higher in subjects thatwent on to achieve a CR (median: 3.9%) or PR (median: 1.1%) compared tosubjects that went on to achieve a best overall response (BOR) of SD orPD (median: 0.51%). Although both CD4⁺ and CD8⁺ CAR T cells wereobserved to have infiltrated the tumor area at the post-treatment timepoint (7 to 20 days after administration), subjects that went on toachieve a CR were observed to have higher ratio of CD8⁺ CAR⁺ T cells toCD4⁺ CAR⁺ T cells, at this post-treatment timepoint, as compared tosubjects that went on to achieve a BOR of SD or PD (CR median: 0.83; SD,PD median: 0.14; p=0.0097).

Comparing matched pre- and post-treatment biopsies from individualsubjects, results showed a trend towards subjects ultimately achieving aBOR of CR or PR having a larger post-treatment increase in CD8⁺ cells(CAR⁺ T and non-CAR T) in tumors, as compared to subjects ultimatelyachieving a BOR of SD or PD (CR, PR median change: ⁺5.3%; SD, PD medianchange: ⁺0.06%; p=0.1225).

Expression of immunosuppressive factors, including CD73, FOXP3, CD163,IDO and PD-L1, varied among subjects at pre-treatment (CD73 (median:1.5%; range 0-42%), FOXP3 (0.10%; 0-1.5%), IDO (0.06%; 0-11%), CD163(1.2%; 0-24%) and PD-L1 (0.16%; 0-56%)) and post-treatment (CD73 (1.6%;0-53%), FOXP3 (0.09%; 0-4.3%), IDO (0.28%; 0-15%), CD163 (3.6%; 0-22%)and PD-L1 (3.3%; 0-65%)). Post-treatment increases in CD8⁺ cells inmatched biopsies were observed to be associated with post-treatmentincreases in IDO (R²=0.64) and PD-L1 (R²=0.61) expression. This resultis consistent with a conclusion that infiltration of CD8⁺ CAR⁺ cells atthe time assessed may indicate potential likelihood of achieven a degreeof response or duration of response, and that the presence and/oractivity of such cells may result in upregulation of TME factors.

D. Conclusion

Durable response at month 3 after CAR⁺ T cell administration wasobserved to be associated with higher levels of CD4⁺ cells inpre-treatment tumors. In post-treatment tumor cells, CAR⁺ T cells, bothCD4⁺ and CD8⁺, were observed to infiltrate the tumor and adjacenttissue. ORR was associated with an increase in CAR⁺ T cells in the tumorbiopsy. An increase of CD8⁺ levels in the post-treatment tumor biopsycompared to CD8⁺ levels in the pre-treatment tumor biopsy was associatedwith increased IDO and PD-L1 expression. In some embodiments, therapiestargeting these pathways, such as those administered at the time of orfollowing administration of the CAR-T cells, may enhance one or moretherapeutic outcomes or duration thereof following CAR⁺ T celladministration.

Example 6: Further Assessment of Response and Safety Outcomes inSubjects with Relapsed and Refractory Non-Hodgkin's Lymphoma (NHL) AfterAdministration of Anti-CD19 CAR-Expressing Cells

Response and safety outcomes were assessed in patients at a subsequentpoint in time in the clinical study described in Examples 1 and 3 above.

A. Subjects and Treatment

The analysis at this time point presented in this example is based onassessment of a total of 102 subjects in the FULL cohort (73 in the COREcohort) that had been administered the anti-CD19 CAR-expressing cells.The FULL cohort included subjects who had DLBCL (DLBCL, NOS de novo andtransformed from follicular lymphoma; high grade B-cell lymphoma(double/triple hit); DLBCL transformed from CLL or MZL; PMBCL; and FL3B,ECOG 0-2, after 2 lines of therapy; the CORE cohort for analysisincluded subjects having DLBCL, NOS and transformed from follicularlymphoma (tFL) or high grade B-cell lymphoma (double/triple hit) andwith Eastern Cooperative Oncology Group performance status (ECOG PS) of0 or 1. Approximately 90% of treated patients in the FULL and the COREcohort had at least one poor-risk disease feature predictive of shortmedian overall survival (OS) of 3-6 months (see Crump et al., Blood(2017) 130:1800-1808 and Van de Neste et al., Bone Marrow Transplant.(2016) 51(1):51-7), such as double/triple hit expressors, primaryrefractory disease, refractory to 2 or more lines of therapy, neverachieved CR, never received autologous stem cell transplant (ASCT) or anECOG PS of 2.

At this time point, a total of 134 subjects had been leukapheresed, ofwhich 2 had compositions unavailable. Product was available for 99% ofapheresed subjects (132/134) in the DLBCL cohort. Of another 18 subjectswhose products were available, 5 had withdrawn, and 13 had developedprogressive disease or had died. A total of 114 subjects had beenadministered the anti-CD19 CAR-expressing cells, of which 12 receivednon-conforming anti-CD19 CAR-expressing cells (compositions notnecessarily meeting certain specifications but deemed to be safe foradministration). Subjects had received DL1 (n=45), double dose of DL1(n=6) or DL2 (n=51). Seven (7) subjects with mantle cell lymphoma (MCL)had been administered CAR⁺ cells at DL1. At this time point, eight (8)subjects were treated in an outpatient setting.

The demographics and baseline characteristics of the FULL and COREcohort subjects at the timepoint are set forth in Table E18.

TABLE E18 Patient Characteristics: DLBCL Cohort Characteristic FULL (n =102) CORE (n = 73) Median age (range), years 61 (20-82) 60 (20-82) ≥0(20-82) (range 37 (36) 24 (33) B-NHL Subtype, n (%) DLBCL, NOS de novo63 (62) 53 (73) Transformed from FL (tFL) 23 (23) 20 (27) Transformedfrom MZL 6 (6)/6 (6) 0 (tMZL)/CLL (tCLL) Follicular, grade 3B/PMBCL 1(1)/3 (3) 0 Molecular Subtype, n (%) Double/triple hit 19 (19) 16 (22)Patient Characteristics, n (%)L ECOGPS 0-1 93 (91)  73 (100) IPI 3-5 43(42) 26 (36) CNS involvement 2 (2) 1 (1) Chemorefractory^(b) 71 (70) 49(67) Prior lines of therapy, 3 (1-8) 3 (2-8) median (range) Neverachieved CR 49 (48) 36 (49) Any HSCT 41 (40) 28 (38) Prior autologous 38(37) 28 (38) Prior allogeneic 5 (5) 0 HSCT, hematopoietic stem celltransplant. IPI, International Prognostic Index; SD, stable disease;WHO, World Health Organization. ^(a)At trial initiation, included inDLBCL, NOS histology; based on most recent WHO criteria (Swerdlow etal., (2016) Blood 127(20): 2375-2390), are now considered high-gradeB-cell lymphoma, with myc and bcl2 and/or bcl6 rearrangements with DLBCLhistology (double/triple hit). ^(b)SD or PD to lastchemotherapy-containing regimen or relapse <12 months after autologousSCT.

B. Safety and Response Outcomes after Treatment

Table E19 shows the safety outcome of the FULL and CORE cohort. Asshown, no deaths from CRS or NT were observed. In the FULL cohort, themedian time to onset of CRS was 5 days (range, 2-12 days) and NT was 10days (range, 3-23 days). In the FULL cohort, 17% (n=17) receivedtocilizumab and 21% (n=21) received corticosteroids as a toxicityintervention. In the CORE cohort, no increase in CRS or NT was observedat DL2, compared to DL1.

TABLE E19 Safety OutcomesAfter CAR⁺ Cell Administration FULL CORE AllDose All Dose^(a) Levels Levels DL1S DL2S n = 102 n = 73 n = 33 n = 37CRS, n (%) Any grade 38 (37) 27 (37) 14 (42) 11 (30) Grade 1/2 37 (36)26 (36) 13 (39) 11 (30) Grade 3/4 (sCRS) 1 (1) 1 (1) 1 (3) 0Neurotoxicity, n (%) Any grade 23 (23) 18 (25)  8 (24)  9 (24) Grade 1/210 (10)  7 (10) 1 (3)  6 (16) Grade 3/4 (sNT) 13 (13) 11 (15)  7 (21) 3(8) Any, n (%) CRS or NT 44 (43) 32 (44) 15 (45) 15 (41) sCRS or sNT 13(13) 11 (15)  7 (21) 3 (8) ^(a)Three patients treated on DL1D (doselevel 1, two-dose schedule) with similar outcomes.

FIG. 29 depicts the percentage of subjects in the FULL cohort at thistimepoint (n=102) who were observed to have experienced laboratoryabnormalities and treatment-emergent adverse events (TEAEs) (data for 6subjects with MCL treated with conforming product at DL1 with at least28 days of follow-up are not included; showing TEAEs and laboratoryabnormalities occurring in 20% or more of the subjects).

As shown in Table E20, high rates of response was observed in subjectswith relapsed or refractory (R/R) DLBCL. The results are consistent witha dose response effect on treatment outcome in the CORE cohort. Subjectswith a tumor burden above a threshold (as indicated by the volumetrictumor measurement of sum of product dimensions (SPD) of more than 50cm²) was similarly distributed between subjects receiving DL1 and DL2(approximately 1/3 of the subjects in each group).

TABLE E20 Response After CAR⁺ Cell Administration FULL CORE All Dose AllDose^(a) Levels Levels DL1S DL2S (n = 102) (n = 73) (n = 33) (n = 37)ORR (95% CI), % 75 (65-83) 80 (68-88) 79 (61-91) 78 (62-90) CR (95% CI),% 55 (45-65) 59 (47-70) 55 (36-72) 62 (45-78) 3-mo ORR (95% CI), % 51(41-61) 59 (47-70) 52 (34-69) 65 (48-80) 3-mo CR (95% CI), % 38 (29-48)45 (34-57) 36 (20-55) 51 (34-68) 6-mo ORR (95% CI), % 40 (31-50) 47(35-59) 42 (26-61) 49 (32-66) 6-mo CR (95% CI), % 34 (25-44) 41 (30-53)33 (18-52) 46 (30-63) ^(a)Three patients treated on DL1D (dose level 1,two-dose schedule) with similar outcomes.

Six-month objective response rates (ORR) among various subgroups ofsubjects in the poor-risk DLBCL subgroups, that included all DLBCLpatients treated at all dose levels in the CORE cohort, are shown inFIG. 30 . The results showed high durable ORR in the poor-risk DLBCLsubgroup for anti-CD19 CAR⁺ T cell administration.

Results for the duration of response (DOR, with median follow-up of 8months) and overall survival (grouped by best overall response(non-responder, CR/PR, CR and/or PR), with median follow-up of 12months) are shown for the full cohort and the core cohort cohorts ofsubjects, in FIGS. 31A-31D. The results showed that in the CORE cohort,88% of subjects with CR at 3 months continued to show CR at 6 months,and 93% of subjects who exhibited CR at 6 months continued to show aresponse longer term.

The results were consistent with an observation that administration ofanti-CD19 CAR+ cell compositions that contains a precise and consistentdose of CD4⁺ and CD8⁺ CAR+ T cells results in durable response insubjects with R/R aggressive NHL with poor prognosis and/or heavypretreatment. The results showed a favorable durable response rate inthe CORE cohort, with 49% ORR and 46% CR rate at 6 months, and 93% ofthe subjects (at all dose levels) in CR at 6 months remained in responseat this time point. The results also were consistent with manageabletoxicity and a favorable safety profile, including low rates of severeCRS (1%) and severe neurotoxicity (13%), which, in some aspects,supports outpatient administration.

The present invention is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the invention. Various modifications tothe compositions and methods described will become apparent from thedescription and teachings herein. Such variations may be practicedwithout departing from the true scope and spirit of the disclosure andare intended to fall within the scope of the present disclosure.

SEQUENCES

SEQ ID NO. SEQUENCE DESCRIPTION 1 ESKYGPPCPPCP spacer (IgG4hinge) (aa)Homo sapiens 2 GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT spacer (IgG4hinge)(nt) homo sapiens 3ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE Hinge-CH3SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHY spacerTQKSLSLSLGK Homo sapiens 4ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE Hinge-CH2-VQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK CH3 spacerGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE Homo sapiensWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHN HYTQKSLSLSLGK 5RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQE IgD-hinge-FcERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEV Homo sapiensAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNA SRSLEVSYVTDH 6LEGGGEGRGSLLTCGDVEENPGPR T2A artificial 7MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSI tEGFRSGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHA artificialFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM 8 FWVLVVVGGVLACYSLLVTVAFIIFWVCD28 (amino acids 153- 179 of Accession No. P10747) Homo sapiens 9IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYS CD28 (aminoLLVTVAFIIFWV acids 114- 179 of Accession No. P10747) Homo sapiens 10RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS CD28 (amino acids 180- 220 ofP10747) Homo sapiens 11 RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSCD28 (LL to GG) Homo sapiens 12KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 4-1BB (amino acids 214-255 of Q07011.1) Homo sapiens 13RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP CD3 zetaQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA Homo sapiens LPPR14 RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP CD3 zetaQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA Homo sapiens LPPR15 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP CD3 zetaQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA Homo sapiens LPPR16 RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPL tEGFRDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVV artificialSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLL LWALGIGLFM 17EGRGSLLTCGDVEENPGP T2A artificial 18 GSGATNFSLLKQAGDVEENPGP P2A 19ATNFSLLKQAGDVEENPGP P2A 20 QCTNYALLKLAGDVESNPGP E2A 21VKQTLNFDLLKLAGDVESNPGP F2A 22PGGG-(SGGGG)5-P- wherein P is proline, G is glycine linkerand S is serine 23 GSADDAKKDAAKKDGKS Linker 24 GSTSGSGKPGSGEGSTKG Linker25 gacatccagatgacccagaccacctccagcctgagcgccagcctgggcgaccgg Sequencegtgaccatcagctgccgggccagccaggacatcagcaagtacctgaactggtat encodingcagcagaagcccgacggcaccgtcaagctgctgatctaccacaccagccggctg scFvcacagcggcgtgcccagccggtttagcggcagcggctccggcaccgaetacagectgaccatctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaacacactgccctacacctttggcggcggaacaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcgagggcagcaccaagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcctgagcgtgacctgcaccgtgagcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatggctgggcgtgatctggggcagcgagaccacctactacaacagcgccctgaagagccggctgaccatcatcaaggacaacagcaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcaccagcgtgaccgtgagcagc 26 X₁PPX₂P HingeX₁ is glycine, cysteine or arginine X₂ is cysteine or threonine 27Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Hinge Cys Pro 28Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Hinge 29ELKTPLGDTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTP Hinge PPCPRCP 30Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Hinge 31Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 32Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 33Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Hinge 34Glu Val Val Val Lys Tyr Gly Pro Pro Cys Pro Pro Cys Hinge Pro 35RASQDISKYLN FMC63 CDR L1 36 SRLHSGV FMC63 CDR L2 37 GNTLPYTFGFMC63 CDR L3 38 DYGVS FMC63 CDR H1 39 VIWGSETTYYNSALKS FMC63 CDR H2 40YAMDYWG FMC63 CDR H3 41EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS FMC63 VHETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMD YWGQGTSVTVSS 42DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRL FMC63 VLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT 43DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRL FMC63 scFvHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 44 KASQNVGTNVA SJ25C1 CDR L1 45 SATYRNSSJ25C1 CDR L2 46 QQYNRYPYT SJ25C1 CDR L3 47 SYWMN SJ25C1 CDR Hl 48QIYPGDGDTNYNGKFKG SJ25C1 CDR H2 49 KTISSWDFYFDY SJ25C1 CDR H3 50EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPG SJ25C1 VHDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFY FDYWGQGTTVTVSS 51DIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYR SJ25C1 VLNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR 52GGGGSGGGGSGGGGS Linker 53EVKLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPG SJ25C1 scFvDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCARKTISSVVDFYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSGTDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKR 54 HYYYGGSYAMDY FMC63 HC- CDR3 55 HTSRLHSFMC63 LC- CDR2 56 QQGNTLPYT FMC63 LC- CDR3 57 ACACGGCCTCGTGTATTACTGTIGH primer 58 ACCTGAGGAGACGGTGACC IGH Primer

1-155. (canceled)
 156. A method of treating a subject having smalllymphocytic lymphoma (SLL), the method comprising administering to thesubject a dose of T cells comprising T cells expressing a chimericantigen receptor (CAR) that specifically binds to CD19, the dose of Tcells comprising a ratio of CD4⁺ cells expressing the CAR to CD8⁺ cellsexpressing the CAR of between at or about 5:1 and at or about 1:5,wherein the subject has relapsed following remission after treatmentwith, or become refractory to, one or more prior therapies, wherein theCAR comprises an scFv specific for CD19, a transmembrane domain, acytoplasmic signaling domain derived from a costimulatory molecule, acytoplasmic signaling domain that comprises a CD3zeta signaling domain,and a spacer between the transmembrane domain and the scFv.
 157. Themethod of claim 156, wherein the subject has relapsed followingremission after treatment with, or become refractory to, two or moreprior lines of therapy.
 158. The method of claim 156, wherein thesubject has relapsed following remission after treatment with, or becomerefractory to, one prior therapy.
 159. The method of claim 156, whereinthe dose of T cells comprises between at or about 5×10⁷ CAR-expressing Tcells and 1×10⁸ CAR-expressing T cells.
 160. The method of claim 156,wherein the dose of T cells comprises about 1×10⁸ CAR-expressing T cells161. The method of claim 156, wherein the dose of T cells comprisesadministering a plurality of separate compositions, the plurality ofseparate compositions comprising a first composition comprising one ofthe CD4⁺ T cells and the CD8⁺ T cells and a second compositioncomprising the other of the CD4⁺ T cells and the CD8⁺ T cells.
 162. Themethod of claim 156, wherein the costimulatory molecule is 4-1BB.
 163. Amethod of treating a subject having chronic lymphocytic leukemia (CLL),the method comprising administering to the subject a dose of T cellscomprising T cells expressing a chimeric antigen receptor (CAR) thatspecifically binds to CD19, the dose of T cells comprising a ratio ofCD4⁺ cells expressing the CAR to CD8⁺ cells expressing the CAR ofbetween at or about 5:1 and at or about 1:5, wherein the subject hasrelapsed following remission after treatment with, or become refractoryto, one or more prior therapies wherein the CAR comprises an scFvspecific for CD19, a transmembrane domain, a cytoplasmic signalingdomain derived from a costimulatory molecule, a cytoplasmic signalingdomain that comprises a CD3zeta signaling domain, and a spacer betweenthe transmembrane domain and the scFv.
 164. The method of claim 163,wherein the subject has relapsed following remission after treatmentwith, or become refractory to, two or more prior lines of therapy 165.The method of claim 163, wherein the subject has relapsed followingremission after treatment with, or become refractory to, one priortherapy.
 166. The method of claim 163, wherein the dose of T cellscomprises between at or about 5×10⁷ CAR-expressing T cells and 1×10⁸CAR-expressing T cells.
 167. The method of claim 163, wherein the doseof T cells comprises about 1×10⁸ CAR-expressing T cells.
 168. The methodof claim 163, wherein the dose of T cells comprises administering aplurality of separate compositions, the plurality of separatecompositions comprising a first composition comprising one of the CD4⁺ Tcells and the CD8⁺ T cells and a second composition comprising the otherof the CD4⁺ T cells and the CD8⁺ T cells.
 169. A method of treating asubject having Mantle cell lymphoma (MCL), the method comprisingadministering to the subject a dose of T cells comprising T cellsexpressing a chimeric antigen receptor (CAR) that specifically binds toCD19, the dose of T cells comprising a ratio of CD4⁺ cells expressingthe CAR to CD8⁺ cells expressing the CAR of between at or about 5:1 andat or about 1:5 wherein the subject has relapsed following remissionafter treatment with, or become refractory to, one or more priortherapies wherein the CAR comprises an scFv specific for CD19, atransmembrane domain, a cytoplasmic signaling domain derived from acostimulatory molecule, a cytoplasmic signaling domain that comprises aCD3zeta signaling domain, and a spacer between the transmembrane domainand the scFv.
 170. The method of claim 169, wherein the subject hasrelapsed following remission after treatment with, or become refractoryto, two or more prior lines of therapy.
 171. The method of claim 169,wherein the subject has relapsed following remission after treatmentwith, or become refractory to, one prior therapy.
 172. The method ofclaim 169, wherein the dose of T cells comprises between at or about5×10⁷ CAR-expressing T cells and 1×10⁸ CAR-expressing T cells.
 173. Themethod of claim 169, wherein the dose of T cells comprises about 1×10⁸CAR-expressing T cells.
 174. The method of claim 169, wherein the doseof T cells comprises administering a plurality of separate compositions,the plurality of separate compositions comprising a first compositioncomprising one of the CD4⁺ T cells and the CD8⁺ T cells and a secondcomposition comprising the other of the CD4⁺ T cells and the CD8⁺ Tcells.
 175. The method of claim 169, wherein the costimulatory moleculeis 4-1BB.